C-met receptor regulation by angiotensin iv (at4) receptor ligands

ABSTRACT

The cell surface c-Met receptor, through which hepatocyte growth factor (HGF) signals are mediated, has now been identified as the Angiotensin-IV receptor (AT(4)R) in processes that include HGF-regulated cell motility, angiogenesis cancer metastasis and others. Disclosed are angiotensin-like factor compositions and methods for using them to diagnose, prevent and/or treat conditions associated with c-Met dysregulation, including altering hepatocyte growth factor activity or c-Met receptor activity by administering an angiotensin-like factor that specifically binds to a cell surface c-Met receptor.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 60/819,201 filed Jul. 7, 2006 whichapplication is hereby incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 730138_(—)404_SEQUENCE_LISTING.txt. The textfile is 77 KB, was created on Jul. 6, 2007, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND

1. Technical Field

Embodiments of the invention disclosed herein relate generally tocompositions and methods for altering hepatocyte growth factor activityor c-Met receptor activity. Certain aspects relate to the diagnosis,prevention or treatment of, as well as to general therapy of subjectshaving, suspected of having, or susceptible to, a condition associatedwith c-Met receptor dysregulation. C-Met receptor dysregulation may be acondition in which underactivity, overactivity or improper activity of ac-Met cellular or molecular event is present, including ahyperproliferative disorder, a condition characterized by abnormalangiogenesis, or alternatively, a condition characterized by vascularinsufficiency such as may benefit from increased angiogenesis.Embodiments disclosed herein further relate to methods for identifyingor modifying compounds useful for the diagnosis, prevention or treatmentof such conditions associated with c-Met receptor dysregulation.

2. Description of the Related Art

The classic rennin-angiotensin system regulates cardiovascular functionincluding blood pressure, electrolyte balance, reproduction, and mayplay a role in other physiological processes, including atherosclerosis.These angiotensin-mediated effects are believed primarily to operatethrough angiotensin (AT) receptors identified as AT₁ and AT₂ receptors.Renin, through its proteolytic activity, first cleaves theangiotensinogen precursor polypeptide to form angiotensin I. Next,angiotensin converting enzyme (ACE) enzymatically converts angiotensin Ito angiotensin II; ACE has been detected in a variety of tissuesincluding brain, kidney, adrenal glands, vasculature, heart and ovaries.ACE-generated angiotensin II (AT2 or AT₂) is subsequently cleaved (byaminopeptidase A) to form angiotensin III, which is cleaved byaminopeptidases N, M and/or B to form angiotensin IV(Val-Tyr-Ile-His-Pro-Phe, [SEQ ID NO:33]). (Mustafa et al., J. ReninAngiotens. Aldoster. Syst. 2(4):205-210, (2001), Thomas et al., Int. J.Biochem. & Cell Biol. 35: 774-779 (2003); McKinley et al., Int. J.Biochem. & Cell Biol. 35: 901-918 (2003).)

Angiotensin IV (AT₄ or AT4) has been shown to play a role in regulatingdisparate biological activities including blood flow, cognitivefunction, neuronal development, inflammation and behavior (Wright etal., Prog. Neurobiol. 72: 263-293 (2004); Kamar et al., Regul. Pept. 68:131-138 (1997).) AT4 is believed to exert its biological effects throughinteraction with a cell surface receptor identified as the AT₄ receptor(AT(4)R), which is also known as the insulin-regulated membraneaminopeptidase (IRAP) (e.g., Chai et al., 2004 Cell. Mol. Life. Sci.61:2728; Esteban et al., 2005 Circ. Res. 96:965; Albiston et al., 2001J. Biol. Chem. 276:48623). AT(4)R/IRAP is a type II (see, e.g., Parks,1996 J. Biol. Chem. 271:7187) integral membrane-spanning protein havingaminopeptidase activity.

Many of the observed biological characteristics of the AT₄ system (e.g.,the existence of both agonist and antagonist AT4 ligands), however, havebeen difficult to reconcile with the IRAP model for the AT₄ receptor.This discrepancy suggested that another unidentified protein(s) might beresponsible for the action of AT₄ receptor ligands (Harding et al., 1994Kidney Int. 46:1510; Wright et al., 2004 Prog. Neurobiol. 72:263). Themolecular identity of the target AT₄ receptor that mediates a number ofAT4 biological activities has, however, remained elusive.

Hepatocyte growth factor (HGF, e.g., 1991 Proc. Nat. Acad. Sci. USA88:7001; GenBank Accession No. AAA64239 [SEQ ID NO:83], M73239.1 (728amino acids)), also known as scatter factor (SF), is a growth factorthat induces cell motility and cell proliferation, which may lead tonormal processes of angiogenesis, or abnormal processes of tumordevelopment or metastasis. HGF functions by binding to its cell surfacereceptor, c-Met, which is a receptor protein tyrosine kinase and aprotooncogene product.

The c-Met receptor is a heterodimer composed of an alpha and beta chain(Maggiora et al., J. Cell Physiol. 173:183-186, (1997), Christensen etal., Can. Lett. 225: 1-26, (2005)). The c-Met receptor is enriched onvascular endothelial cells where it mediates the regulation ofangiogenesis (Rosen et al., 1997 EXS 79:193). For instance, NK4, a largemolecule c-Met inhibitor, has been shown previously to inhibitangiogenesis (Kuba et al., 2000 Cancer Res. 60:6737).

Upon activation, as may result from ligand engagement, the c-Metpolypeptide (e.g., hepatocyte growth factor-receptor, HGF-R, also knownas scatter factor receptor, SF-R, GenBank Acc. No. AAA59591, [SEQ IDNO:84]) induces mitogenic, motogenic and morphogenic responses byrecruiting a number of signaling and docking molecules, and has beenimplicated in the phosphorylation of cell junction proteins (e.g., Zhanget al., 2003 J. Cell Biochem. 88:408; Miao et al., 2003 J. Cell Biol.162:1281; Berdichevsky et al., 1994 J. Cell Sci. 107:3557). Ligandinduced activation of c-Met by HGF/SF leads to the autophosphorylationof specific tyrosine residues within the c-Met receptor protein tyrosinekinase (PTK) domain (Furge et al. (2000) Oncogene 19, 5582-5589; Weidneret al. (1995) Proc Natl Acad Sci USA 92, 2597-2601) and to theassociation of various signaling proteins (e.g., Naldini et al., 1991Mol. Cell. Biol. 1250:1085). A significant event in c-Met signaling isthe association with the c-met receptor of growth factor receptor boundprotein 2, Grb2 associated binder (Gab1), a multi-functional scaffoldingadapter (Birchmeier et al., 2003 Nat. Rev. Mol. Cell. Biol. 4:915). Gab1association provides c-Met with multiple docking sites for a variety ofintracellular signal transducers (Trusolino et al., 2002 Nat. Rev.Cancer 2:289).

Following activation by HGF/SF, c-Met is able to exert a variety ofeffects by recruiting docking and signaling molecules. Phosphorylationof the tyrosine residues in the activation loop of the PTK domainpotentiates the intrinsic kinase activity of Met, whereasphosphorylation of the two docking site tyrosine residues (Tyr¹³⁴⁹,Tyr¹³⁵⁶) allows for the recruitment of adaptor molecules including Grb2,SHC and Gab 1 and signaling enzymes including phosphotidylinositol3-kinase (PI3K), phospholipase Cγ (PLC-γ), the PTK src, the proteintyrosine phosphatase SHP2, as well as the transcription factor STAT3(reviewed in Furge et al. (2000) Oncogene 19, 5582-5589).

The binding of HGF to the cell surface c-Met receptor thus results inmultiple cell-signaling events that promote cell survival, cellproliferation, cell motility, disruption of the extracellular matrix(ECM), cell morphogenesis, angiogenesis and/or cell extravasation andcolonization, for instance, as observed in tumor metastasis. (Jeffers etal., J. Mol. Med., 74: 505-513 (1996); Amicone et al., EMBO J. 16:495-503 (1997); Matsumoto and Nakamura, Biochem. Biophys. Res. Comm.239: 639-644 (1997); Kirchhofer et al., J. Biol. Chem., 279: 39915-39924(2004)). Disruption of normal signaling through c-Met has beenimplicated in certain cancers (e.g., Zhang et al., 2004 Cancer Cell 6:5;Christensen et al., 2005 Cancer Lett. 225:1-26; Ferraro et al., 2006Oncogene 25:3689). For example, overexpression of HGF and/or of c-Methas been implicated in a number of cancers, including carcinomas,gliomas, and mesotheliomas. Particular organs affected include breast,pancreas, liver, lung, ovary, stomach, bile duct, kidney, and others, inpart because of increased angiogenesis (Zbar et al., J. Urol., 151:561-566 (1994); Date et al., FEBS Letters, 420:1-6 (1997)). In addition,several studies have indicated that cancer cells can be a significantsource of HGF within a subject (e.g., Jiang et al., Onc. Hemat. 53:35-69 (2005)).

Alterations (e.g., statistically significant increases or decreases) inthe activity states of a number of intracellular signaling cascades thuscharacterize cellular responses to HGF binding by the cell surface c-Metreceptor, including biological signal transduction pathways thatcomprise one or more of Grb2, cortactin, Arp2/3, WASP/Wave, Rho/rac,Rock, LIMK, PI1P5-K, ERM proteins, Dia-1, MLC phosphatase, cofilin,Ptdins(4,5)P2, cadherins (including E-cadherin), MMPs, fl-catenin,p27^(kiP1), SOS, Ras, Raf, MAPK, PI3K, NK B, src, JNK1, Bid/Bax,caspases, C-Myc, Bax, Mcl1, Bcl-w, Akt, FLICE, STATs (including STAT3),COX, ERK/paxillin, as well as others (see, e.g., Jiang et al., Onc.Hemat. 53: 35-69 (2005); Alberts et al., Molecular Biology of the Cell,4^(th) Ed., 2002, Garland Science, NY). Activation of theseintracellular messenger systems can lead to changes in a cell'scytoskeleton, adhesion state and adherens junctions, cell cycle, anddirectional cell movement, and may also contribute to altered activityin one or more of a number of other biochemical pathways that affectcellular metabolic, catabolic, biosynthetic, respiratory, geneexpression, membrane dynamic or other functions or phenotypes. Thus,such HGF-c-Met binding events may lead to or contribute to cancerdevelopment, tumor cell growth or metastasis, altered angiogenesis, orother physiologically significant outcomes.

Angiogenesis, the process of blood vessel formation, is necessary forproper wound healing and repair, as well as playing an important roleduring embryonic, fetal and young animal development, and continuing onto adulthood. Dysfunction in the course of angiogenic processes at anyof these stages may result in certain detrimental health conditions,including, for instance, ischemic heart disease, preeclampsia,neurodegeneration, and/or respiratory distress (e.g., as the result ofan inadequate or insufficient level of angiogenesis relative to thelevels seen in unafflicted individuals), and also including, forexample, malignant metastasis, arthritis, macular degeneration, diabeticretinopathy, ocular and inflammatory disorders, obesity, asthma,diabetes, cirrhosis, multiple sclerosis, endometriosis, AIDS, bacterialinfections, and/or autoimmune diseases (e.g., as the result of anexcessive or overabundant level of angiogenesis relative to the levelsseen in unafflicted individuals). See, e.g., Carmeliet, Nature438:932-936 (2005). Intervention to alter (e.g., increase or decrease ina statistically significant manner) angiogenesis in these and otherconditions remains a useful but incompletely fulfilled goal.

Clearly there is a need for improved compositions and methods toregulate cellular processes such as cell proliferation, cell motilityand cell survival, that have clinical relevance to cancer, inflammatorydisease, and other conditions. By advancing the understanding ofbiological signal transduction mechanisms that operate through the c-Metreceptor, the present invention provides such compositions and methodsand offers other related advantages.

BRIEF SUMMARY OF THE EMBODIMENTS

According to certain embodiments of the present invention, there isprovided a method for altering a hepatocyte growth factor activity or ac-Met receptor activity in one cell or in a plurality of cells,comprising contacting (i) a cell or plurality of cells with (ii) acomposition that comprises an isolated angiotensin-like factor, underconditions and for a time sufficient for the angiotensin-like factor tointeract with a cell surface c-Met receptor, wherein theangiotensin-like factor is capable of specifically binding to the cellsurface c-Met receptor, and thereby altering hepatocyte growth factoractivity or c-Met receptor activity. In certain further embodiments theangiotensin-like factor comprises a composition that is selected from(a) a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof,of general formula I: N—X₁—X₂—X₃—C [I] wherein N is an amino terminus ofthe peptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, C is a carboxyterminus of the peptide or peptidomimetic and consists of 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids, X₁ isphenylalanine, tryptophan or tyrosine, X₂ is isoleucine, leucine,alanine, valine, phenylalanine, proline, methionine or tryptophan, andX₃ is lysine, arginine or histidine; (b) a polypeptide of no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, of general formula I: N—X₁—X₂—X₃—C[I] wherein N is an amino terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, C is a carboxy terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, X₁ is a natural or non-naturalamino acid having an aromatic side chain, X₂ is a natural or non-naturalamino acid having a hydrophobic side chain, and X₃ is a natural ornon-natural amino acid having a basic side chain; (c) a polypeptide ofno more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4 or 3 amino acids, or a peptidomimetic thereof, said polypeptidecomprising (i) a tripeptide having an amino acid sequence that isselected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and (ii) atleast one of an amino terminus and a carboxy terminus, each of saidamino terminus and said carboxy terminus consisting of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; (d) thepolypeptide or peptidomimetic of (c) which comprises the amino acidsequence Nle-Tyr-Leu-Ψ-His-Pro-Phe as set forth in SEQ ID NO:43, whereinΨ consists of a reduced peptide bond of formula II: —CH₂—NH₂— [II], (e)the polypeptide or peptidomimetic of (b) which comprises at least oneamino acid sequence selected from the group consisting ofAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO:9),Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO:25), Val-Tyr-Ile-His-Pro-Phe (SEQID NO:33), Nle-Tyr-Ile-His-Pro-Phe (SEQ ID NO:41),Val-Ψ-Tyr-Leu-Ψ-His-Pro-Phe (SEQ ID NO:42), Nle-Ψ-Tyr-Ile-His-Pro-Phe(SEQ ID NO:45), Leu-Ψ-Tyr-Leu-Ψ-His-Pro-Phe (SEQ ID NO:46),Nle-Tyr-Ile-His (SEQ ID NO:47), Nle-Tyr-Ile-(CH₂)₆-Phe-amide (SEQ IDNO:48), Nle-Tyr-Ile-Sar-Sar-dPhe (SEQ ID NO:49),Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO:50),Nle-Tyr-Ile-6(amino) hexanoic acid amide (SEQ ID NO:52),Nle-Tyr-Ile-His-Pro (SEQ ID NO:53), Lys-Tyr-Ile-His-Pro-Phe (SEQ IDNO:54), benzyl-cysteine-Tyr-Ile-His-Pro-Phe (SEQ ID NO:55),dNle-Tyr-Ile-His-Pro-Phe (SEQ ID NO:56), Nle-Tyr-Ile-Ψ-His-Pro-Phe [SEQID NO:57], Nle-Tyr-Val-Ψ-His-Pro-Phe [SEQ ID NO:58],Nle-Tyr-Nle-Ψ-His-Pro-Phe [SEQ ID NO:59], Nle-Phe-Leu-Ψ-His-Pro-Phe [SEQID NO:60], Nle-Phe-Ile-Ψ-His-Pro-Phe [SEQ ID NO:61],Nle-Phe-Val-Ψ-His-Pro-Phe [SEQ ID NO:62], Nle-Phe-Nle-Ψ-His-Pro-Phe [SEQID NO:63], Nle-Tyr-Leu-Ψ-Arg-Pro-Phe [SEQ ID NO:64],Nle-Tyr-Ile-Ψ-Arg-Pro-Phe [SEQ ID NO:65], Nle-Tyr-Val-V-Arg-Pro-Phe [SEQID NO:66], Nle-Tyr-Nle-Ψ-Arg-Pro-Phe [SEQ ID NO:67],Nle-Phe-Leu-V-Arg-Pro-Phe [SEQ ID NO:68], Nle-Phe-Ile-Ψ-Arg-Pro-Phe [SEQID NO:69], Nle-Phe-Val-Ψ-Arg-Pro-Phe [SEQ ID NO:70],Nle-Phe-Nle-Ψ-Arg-Pro-Phe [SEQ ID NO:71], Nle-Tyr-Leu-Ψ-Lys-Pro-Phe

[SEQ ID NO:72], Nle-Tyr-Ile-Ψ-Lys-Pro-Phe [SEQ ID NO:73],Nle-Tyr-Val-Ψ-Lys-Pro-Phe [SEQ ID NO:74], Nle-Tyr-Nle-Ψ-Lys-Pro-Phe [SEQID NO:75], Nle-Phe-Leu-V-Lys-Pro-Phe [SEQ ID NO:76],Nle-Phe-Ile-Ψ-Lys-Pro-Phe [SEQ ID NO:77], Nle-Phe-Val-Ψ-Lys-Pro-Phe [SEQID NO:78], Nle-Phe-Nle-Ψ-Lys-Pro-Phe [SEQ ID NO:79], γ-aminobutyricacid-Tyr-Ile [SEQ ID NO:80], and β-Ala-Tyr-Ile [SEQ ID NO:81], wherein Ψconsists of a reduced peptide bond of formula II: —CH₂—NH₂— [II].

In certain other further embodiments the angiotensin-like factorcomprises an antibody, or an antigen-binding fragment of said antibody,that competitively inhibits binding of a native HGF hinge regionpolypeptide to the cell surface c-Met receptor, said native HGF hingeregion polypeptide comprising the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82]. In a still furtherembodiment the antibody is a monoclonal antibody. In another embodimentthe antibody is a chimeric antibody or a humanized antibody. In anotherembodiment the antigen-binding fragment is selected from the groupconsisting of a Fab fragment, a Fab′ fragment, a (Fab′)₂ fragment, an Fdfragment, an Fv fragment and an scFv. In another embodiment the antibodycomprises an anti-idiotype antibody that specifically recognizes acomplementarity determining region of an immunoglobulin thatspecifically binds to the native HGF hinge region polypeptide. Incertain other embodiments related to the foregoing embodiments, theangiotensin-like factor competitively inhibits binding of native HGF tothe cell surface c-Met receptor.

In another embodiment the step of contacting is selected from the groupconsisting of contacting in vivo and contacting ex vivo. In otherembodiments the angiotensin-like factor is glycosylated. In anotherembodiment the hepatocyte growth factor activity or the c-Met receptoractivity comprises at least one activity that is selected from the groupconsisting of (i) induction of cellular proliferation, (ii) induction ofcell scattering or migration, and (iii) alteration of a c-Met receptorpathway component phosphorylation state. In a further embodimentalteration of a c-Met receptor pathway component phosphorylation statecomprises induction of Gab1 activation.

In certain related embodiments the angiotensin-like factor comprises apeptidomimetic and the peptidomimetic comprises at least one moiety thatis selected from the group consisting of a non-natural amino acidresidue and a reduced peptide bond. In another embodiment the cell orplurality of cells comprises a tissue or an organ. In another embodimentthe hepatocyte growth factor activity or c-Met receptor activity in thecell is decreased subsequent to the step of contacting, relative to thehepatocyte growth factor activity or c-Met receptor activity in the cellprior to the step of contacting. In a further embodiment the hepatocytegrowth factor activity or c-Met receptor activity comprises an activitythat is selected from the group consisting of (i) induction of cellproliferation, (ii) induction of cell migration, (iii) induction ofextracellular matrix disruption, (iv) induction of dysregulation ofapoptosis, (v) induction of cellular extravasation, (vi) induction ofaltered expression of an adhesion molecule that is selected from thegroup consisting of CD44, catenin, catenin p, catenin δ1, MMP-1, PAI-1,thrombospondin 1, and integrin α-2, and (vii) induction of alteredexpression of an angiogenesis-related molecule that is selected from thegroup consisting of: Adamts 1, CD36, Connective tissue growth factor,Pecam 1, Cxcl 4, restin, Ccl 2, TNF-α, VEGF-α, and VEGF-β. In anotherembodiment the hepatocyte growth factor activity or c-Met receptoractivity is increased in the cell subsequent to the step of contacting,relative to the hepatocyte growth factor activity or c-Met receptoractivity in the cell prior to the step of contacting. In a furtherembodiment the hepatocyte growth factor activity or c-Met receptoractivity comprises an activity that is selected from the groupconsisting of (i) induction of angiogenesis, (ii) induction of neuritegrowth or axon guidance, (iii) induction of cell differentiation, (iv)induction of bone regeneration, and (v) induction of tissue repair.

According to another embodiment of the present invention, there isprovided a method of altering a hepatocyte growth factor activity or ac-Met receptor activity in a subject, comprising administering to thesubject a composition that comprises an isolated angiotensin-likefactor, under conditions and for a time sufficient for theangiotensin-like factor to interact with a cell surface c-Met receptorin the subject, wherein the angiotensin-like factor is capable ofspecifically binding to the cell surface c-Met receptor, and therebyaltering a hepatocyte growth factor activity or a c-Met receptoractivity in the subject. In another embodiment there is provided amethod of treating or preventing a condition associated with c-Metdysregulation in a subject, comprising administering to the subject acomposition that comprises an isolated angiotensin-like factor, underconditions and for a time sufficient for the angiotensin-like factor tointeract with a cell surface c-Met receptor in the subject, wherein theangiotensin-like factor is capable of specifically binding to the cellsurface c-Met receptor, and thereby treating or preventing the conditionassociated with c-Met dysregulation. In a further embodiment thecondition associated with c-Met dysregulation is selected from the groupconsisting of (i) cellular hyperproliferation, (ii) inflammation, (iii)an increased level of angiogenesis relative to the level of angiogenesisin a control subject known to be free of a risk for having a conditionassociated with c-Met dysregulation, (iv) an increased level of adiposedeposition relative to the level of adipose deposition in a controlsubject known to be free of a risk for having a condition associatedwith c-Met dysregulation, and (v) cognitive dysfunction. According tocertain further related embodiments, the angiotensin-like factor isselected from the group consisting of (a) a polypeptide of no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, of general formula I: N—X₁—X₂—X₃—C[I] wherein N is an amino terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, C is a carboxy terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, X₁ is phenylalanine, tryptophan ortyrosine, X₂ is isoleucine, leucine, alanine, valine, phenylalanine,proline, methionine or tryptophan, and X₃ is lysine, arginine orhistidine; (b) a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimeticthereof, of general formula I: N—X₁—X₂—X₃—C [I] wherein N is an aminoterminus of the peptide or peptidomimetic and consists of 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids, C is acarboxy terminus of the peptide or peptidomimetic and consists of 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acidsthat are independently selected from natural and non-natural aminoacids, X₁ is a natural or non-natural amino acid having an aromatic sidechain, X₂ is a natural or non-natural amino acid having a hydrophobicside chain, and X₃ is a natural or non-natural amino acid having a basicside chain; (c) a polypeptide of no more than 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, said polypeptide comprising (i) a tripeptidehaving an amino acid sequence that is selected from the group consistingof: Lys-Asp-Tyr, Leu-Asp-Tyr, Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, andArg-Asn-Cys, and (ii) at least one of an amino terminus and a carboxyterminus, each of said amino terminus and said carboxy terminusconsisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16or 17 amino acids that are independently selected from natural andnon-natural amino acids; (d) the polypeptide or peptidomimetic of (c)which comprises the amino acid sequence Nle-Tyr-Leu-Ψ-His-Pro-Phe as setforth in SEQ ID NO:43, wherein Ψ consists of a reduced peptide bond offormula II: —CH₂—NH₂— [II]; and (e) the polypeptide or peptidomimetic of(b) which comprises at least one amino acid sequence selected from thegroup consisting of SEQ ID NOS:9, 25, 33, 41, 42, 45-50, 52-80 and 81.In certain further embodiments of the foregoing methods, theangiotensin-like factor comprises an antibody, or an antigen-bindingfragment of said antibody, that competitively inhibits binding of anative HGF hinge region polypeptide to the c-Met receptor, said nativeHGF hinge region polypeptide comprising the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82]. In a related embodiment theantibody is a monoclonal antibody. In another related embodiment theantibody is a chimeric antibody or a humanized antibody. In otherrelated embodiments the antigen-binding fragment is selected from thegroup consisting of a Fab fragment, a Fab′ fragment, a (Fab′)₂ fragment,an Fd fragment, an Fv fragment and an scFv. In another embodiment theantibody comprises an anti-idiotype antibody that specificallyrecognizes a complementarity determining region of an immunoglobulinthat specifically binds to the native HGF hinge region polypeptide. Inanother embodiment the angiotensin-like factor competitively inhibitsbinding of native HGF to the c-Met receptor. In another embodiment theangiotensin-like factor is glycosylated. In another embodiment themethod further comprises administering to the subject, in addition tothe angiotensin-like factor, a second composition that comprises atherapeutic agent. In certain further embodiments the therapeutic agentis selected from the group consisting of (a) a chemotherapeutic agent,(b) a nucleic acid disrupting agent, (c) an anti-proliferative agent,(d) a radiotherapy compound, (e) a cytotoxic agent, (f) ananti-inflammatory agent, (g) a statin, (h) a pro-angiogenesis agent, (i)a chemoattractant, and (j) any combination of two or more of (a)-(i).

In certain other embodiments the present invention provides a method ofdiagnosing a subject having, suspected of having or susceptible to acondition associated with c-Met receptor dysregulation, comprising: (a)contacting (i) a first biological sample from a first subject having,suspected of having or susceptible to a condition associated with c-Metreceptor dysregulation, which sample comprises a cell surface c-Metreceptor, and (ii) an isolated angiotensin-like factor, under conditionsand for a time sufficient for the angiotensin-like factor to interactwith the cell surface c-Met receptor, wherein the angiotensin-likefactor is capable of specifically binding to the cell surface c-Metreceptor; and (b) determining an altered level of cell surface c-Metreceptor in the first biological sample, relative to the level of cellsurface c-Met receptor in a second biological sample obtained from asecond subject known to be free of risk for having the conditionassociated with c-Met receptor dysregulation. In certain furtherembodiments the angiotensin-like factor is selected from the groupconsisting of: (a) a polypeptide of no more than 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, of general formula I: N—X₁-X₂—X₃—C [I] wherein Nis an amino terminus of the peptide or peptidomimetic and consists of 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acidsthat are independently selected from natural and non-natural aminoacids, C is a carboxy terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, X₁ is phenylalanine, tryptophan or tyrosine, X₂is isoleucine, leucine, alanine, valine, phenylalanine, proline,methionine or tryptophan, and X₃ is lysine, arginine or histidine; (b) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, ofgeneral formula I: N—X₁—X₂—X₃—C [I] wherein N is an amino terminus ofthe peptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, C is a carboxyterminus of the peptide or peptidomimetic and consists of 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids, X₁ is anatural or non-natural amino acid having an aromatic side chain, X₂ is anatural or non-natural amino acid having a hydrophobic side chain, andX₃ is a natural or non-natural amino acid having a basic side chain; (c)a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, saidpolypeptide comprising (i) a tripeptide having an amino acid sequencethat is selected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and (ii) atleast one of an amino terminus and a carboxy terminus, each of saidamino terminus and said carboxy terminus consisting of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; (d) thepolypeptide or peptidomimetic of (c) which comprises the amino acidsequence Nle-Tyr-Leu-Ψ-His-Pro-Phe as set forth in SEQ ID NO:43, whereinΨ consists of a reduced peptide bond of formula II: —CH₂—NH₂— [II]; and(e) the polypeptide or peptidomimetic of (b) which comprises at leastone amino acid sequence selected from the group consisting of SEQ IDNOS:9, 25, 33, 41, 42, 45-50, 52-80 and 81. In certain relatedembodiments the angiotensin-like factor comprises an antibody, or anantigen-binding fragment of said antibody, that competitively inhibitsbinding of a native HGF hinge region polypeptide to the c-Met receptor,said native HGF hinge region polypeptide comprising the amino acidsequence Lys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82]. In a furtherembodiment the antibody is a monoclonal antibody, or a chimeric antibodyor a humanized antibody. In another embodiment the antigen-bindingfragment is selected from the group consisting of a Fab fragment, a Fab′fragment, a (Fab′)₂ fragment, an Fd fragment, an Fv fragment and anscFv. In another embodiment the antibody comprises an anti-idiotypeantibody that specifically recognizes a complementarity determiningregion of an immunoglobulin that specifically binds to the native HGFhinge region polypeptide. In another embodiment the angiotensin-likefactor is glycosylated. In another embodiment the condition associatedwith c-Met dysregulation is selected from the group consisting ofcellular hyperproliferation, inflammation, reduced angiogenesis, adiposedeposition and cognitive dysfunction.

According to certain other embodiments of the invention there isprovided an antibody, or an antigen-binding fragment of said antibody,that competitively inhibits binding of a native hepatocyte growth factor(HGF) hinge region polypeptide to the c-Met receptor, said native HGFhinge region polypeptide comprising the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82]. In a further embodiment theantibody is a monoclonal antibody, or a chimeric antibody or a humanizedantibody. In another embodiment the antigen-binding fragment is selectedfrom the group consisting of a Fab fragment, a Fab′ fragment, a (Fab′)₂fragment, an Fd fragment, an Fv fragment and an scFv. In anotherembodiment the antibody comprises an anti-idiotype antibody thatspecifically recognizes a complementarity determining region of animmunoglobulin that specifically binds to the native HGF hinge regionpolypeptide. In another embodiment the antibody is capable of alteringat least one HGF activity or c-Met receptor activity. In certain furtherembodiments the hepatocyte growth factor activity or c-Met receptoractivity comprises at least one activity that is selected from the groupconsisting of (i) induction of cellular proliferation, (ii) induction ofcell scattering or migration, and (iii) alteration of a c-Met receptorpathway component phosphorylation state. In certain other embodimentsalteration of a c-Met receptor pathway component phosphorylation statecomprises induction of Gab1 activation.

The present invention also provides in certain embodiments a compositionthat alters a hepatocyte growth factor activity or a c-Met receptoractivity in a cell or in a plurality of cells, comprising a polypeptideor a peptidomimetic thereof that does not comprise the polypeptidesequence set forth in either one of SEQ ID NOS:43 or 54 and that isselected from the group consisting of: (a) a polypeptide of no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, of general formula I: N—X₁—X₂—X₃—C[I] wherein: N is an amino terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, C is a carboxy terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, X₁ is phenylalanine, tryptophan ortyrosine, X₂ is isoleucine, leucine, alanine, valine, phenylalanine,proline, methionine or tryptophan, and X₃ is lysine, arginine orhistidine; (b) a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimeticthereof, of general formula I: N—X₁—X₂—X₃—C [I] wherein: N is an aminoterminus of the peptide or peptidomimetic and consists of 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids, C is acarboxy terminus of the peptide or peptidomimetic and consists of 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acidsthat are independently selected from natural and non-natural aminoacids, X₁ is a natural or non-natural amino acid having an aromatic sidechain, X₂ is a natural or non-natural amino acid having a hydrophobicside chain, and X₃ is a natural or non-natural amino acid having a basicside chain; (c) a polypeptide of no more than 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, said polypeptide comprising (i) a tripeptidehaving an amino acid sequence that is selected from the group consistingof: Lys-Asp-Tyr, Leu-Asp-Tyr, Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, andArg-Asn-Cys, and (ii) at least one of an amino terminus and a carboxyterminus, each of said amino terminus and said carboxy terminusconsisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16or 17 amino acids that are independently selected from natural andnon-natural amino acids; and (d) the polypeptide or peptidomimetic of(b) which comprises at least one amino acid sequence selected from thegroup consisting of SEQ ID NOS:9, 25, 33, 41, 42, 45-50, 52, 53, 55-80and 81.

In another embodiment there is provided a pharmaceutical compositioncomprising: (a) a polypeptide that comprises an amino acid sequenceselected from the group consisting of the sequence set forth in SEQ IDNO:47 and the sequence set forth in SEQ ID NO:52, or a peptidomimeticthereof; and (b) a pharmaceutically acceptable carrier. In anotherembodiment there is provided a pharmaceutical composition comprising apolypeptide, or a peptidomimetic thereof; and a pharmaceuticallyacceptable carrier, wherein the polypeptide or peptidomimetic does notcomprise the polypeptide sequence set forth in either one of SEQ IDNOS:43 or 54 and is selected from the group consisting of: (a) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, ofgeneral formula I: N—X₁—X₂—X₃—C [I] wherein: N is an amino terminus ofthe peptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, C is a carboxyterminus of the peptide or peptidomimetic and consists of 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids, X₁ isphenylalanine, tryptophan or tyrosine, X₂ is isoleucine, leucine,alanine, valine, phenylalanine, proline, methionine or tryptophan, andX₃ is lysine, arginine or histidine; (b) a polypeptide of no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, of general formula I: N—X₁—X₂—X₃—C[I] wherein: N is an amino terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, C is a carboxy terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, X₁ is a natural or non-naturalamino acid having an aromatic side chain, X₂ is a natural or non-naturalamino acid having a hydrophobic side chain, and X₃ is a natural ornon-natural amino acid having a basic side chain (c) a polypeptide of nomore than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4or 3 amino acids, or a peptidomimetic thereof, said polypeptidecomprising (i) a tripeptide having an amino acid sequence that isselected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and (ii) atleast one of an amino terminus and a carboxy terminus, each of saidamino terminus and said carboxy terminus consisting of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; and (d)the polypeptide or peptidomimetic of (b) which comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NOS:9,25, 33, 41, 42, 45-50, 52, 53, 55-80 and 81.

These and other aspects of the invention will be evident upon referenceto the following detailed description and attached drawings. All of theU.S. patents, U.S. patent application publications, U.S. patentapplications including U.S. Ser. No. 60/819,201 (filed Jul. 7, 2006),foreign patents, foreign patent applications and non-patent publicationsreferred to in this specification and/or listed in the Application DataSheet, are incorporated herein by reference in their entirety, as ifeach was incorporated individually. Aspects of the invention can bemodified, if necessary, to employ concepts of the various patents,applications and publications to provide yet further embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (FIG. 1A) a schematic illustration of HGF, angiostatin andangiotensin homology structures and (FIG. 1B) angiotensin system membersand receptor interactions.

FIG. 2 shows the effects on ¹²⁵I-HGF binding to mouse liver membranes ofunlabeled HGF (FIG. 2A) and (FIG. 2B) norleual (Compound 2, SEQ IDNO:43). Mouse liver plasma membranes were incubated with 50 pM ¹²⁵I-HGFand the indicated concentrations of HGF or Norleual. Results arepresented as percent of specific ¹²⁵I-HGF binding and error barsindicate ±standard error of mean (SEM). Competition experiments includedquadruplicate data points and each experiment was repeated intriplicate. FIG. 3 shows the effects of unlabeled HGF (FIG. 3A, rightpanel) or unlabeled norleual (FIG. 3A, left panel) on ¹²⁵I-norleual(FIG. 3A, Compound 2, SEQ ID NO:43) binding to HEK293 cells, and ofunlabeled HGF (FIG. 3B, left panel) or unlabeled norleual (FIG. 3B,right panel) on ¹²⁵HGF binding to HEK293 cells. FIG. 3C: ¹²⁵I-Norleualwas bound to HEK membranes in the presence or absence of 1.5 nM HGF(n=8) or 1 μM Norleual (n=6). Total binding is reported as percentcontrol (n=10), (*p<0.001). Error bars indicate ±SD. FIG. 3D shows theeffects of the indicated concentrations of unlabeled Norleual on bindingof ¹²⁵I-Norleual to HEK293 membranes, with data subject to Prism®nonlinear regression analysis (GraphPad Software Inc., San Diego,Calif.) showing significantly better data fit for two-site competitionmodel than for one-site model (F=7.335, P=0.0074). IC₅₀ values forNorleual were calculated as 48 pM and 7.4 nM. Error bars indicate SD anddata points were performed in duplicate. A repeat experiment showedsimilar results (F=7.825 and P=0.0023).

FIG. 4 shows the effects of Norleual (Compound 2, SEQ ID NO:43) onreceptor protein tyrosine phosphorylation in a tyrosine kinase receptorprotein array (FIG. 4A), with quantification of relative receptorprotein tyrosine phosphorylation shown in FIG. 4B: P-HGF-R control,protein from hepatocyte growth factor receptor control; P-HGF-R Compound2, protein from hepatocyte growth factor receptor treated with Compound2; P-VEGF1-R control, protein from VEGF1 receptor control; P-VEGF1-RCompound 2, protein from VEGF1 receptor treated with Compound 2.

FIG. 5 shows that Norleual (Compound 2, SEQ ID NO:43) inhibitedHGF-dependent c-Met phosphorylation and Gab1 association andphosphorylation in vitro. (FIG. 5A) HEK293 cells were treated for 10minutes with HGF at indicated concentrations. Lysates wereimmunoprecipitated (IP) with anti-c-Met antibody (DO-24) andimmunoblotted (IB) with anti-c-Met or anti-phospho-tyrosine (06-427)antibody. (FIG. 5B) HEK293 cells were treated for 10 minutes with HGFand/or Norleual. Lysates were IP with anti-c-Met (DO-24) and IB withanti-c-Met (DQ-13) or anti-phospho-tyrosine (HAM 1676). (FIG. 5C) HEK293lysates were IP with anti-c-Met (DO-24) and IB with anti-Gab1. (FIG. 5D)HEK 293 lysates were IP with anti-Gab1 and IB with anti-Gab1 oranti-phospho-tyrosine (HAM 1676). (FIGS. 5E, 5F, 5G) Relative amounts(normalized to % total c-Met in IP) of quantified IP/IB band intensitiesfor control, 110 pM HGF-, 110 pM HGF and 20 pM Norleual-, and 20 pMNorleual-treated HEK293 cells for phospho-c-Met (FIG. 5E, n=4),phospho-Gab1 (FIG. 5F, n=4), and c-Met associated Gab1 (FIG. 5G, n=3).Error bars indicate ±SD.

FIG. 6 shows Norleual (Compound 2, SEQ ID NO:43) inhibition ofHGF-dependent Gab1 phosphorylation in vitro. HEK293 cells were treatedfor 10 minutes with HGF and/or Norleual at the indicated concentrations.Lysates were IP with anti-c-Met (DO-24, Millipore/Upstate, Billerica,Mass.) and IB with anti-c-Met (DQ-13, Millipore/Upstate) oranti-phosphotyrosine (HAM1676, lot JLB03, R&D Systems, Inc.,Minneapolis, Minn.). Longer film exposures revealed bands with atapproximate molecular weight of 100 kDa, the molecular weight of Gab1.

FIG. 7 illustrates the ability of Compound 2 (SEQ ID NO:43) to inhibitHGF-dependent cell scattering in MDCK cells.

FIG. 8 shows inhibition by Norleual (Compound 2, SEQ ID NO:43) ofHGF-induced cell scattering in MDCK cells. FIG. 8A: Confluent MDCK cellson coverslips were treated with HGF (110 pM), or HGF (110 pM)+Norleual(100 pM) for 48 hours. Digital images of top, bottom, left, and rightsectors were acquired and the scattering of cells In each digital imagewas scored in blinded fashion and normalized to control scores. Thedifference between HGF and HGF+Norleual groups was significant(*p=0.0245, n=3). Error bars indicate ±SEM. FIG. 8B: MDCK cells wereplated on 6 mm diameter coverslips in DMEM 10% FBS and grown to 100%confluency. The coverslips were then transferred to fresh 6 well platesand the cells were serum-deprived for 24 hours to synchronize the cellsin the same growth state. Thereafter, the 20 ng/ml of HGF alone and inthe presence of 10⁻¹⁰ M Norleual in a 1% FBS DMEM was added to thecells. The cells were allowed scatter off of the coverslip for 24 hoursbefore being fixed with 100% methanol for 15 minutes followed bystaining with Diff-Quik (Dade-Behring). The coverslip was then removedto reveal the ring of cells that had scattered off of the coverslip andonto the plate. This ring was photographed and loaded on to NIH image J.Using NIH image J, the pixel count was determined for each ring. One-wayANOVA with tukey's post-hoc analysis was used to determine differencesbetween the treatments (Prism® software, GraphPad, San Diego, Calif.). *indicates p<0.001 vs. HGF treatment. N=4, mean +/−SEM.

FIG. 9 shows inhibition by Norleual of HGF_induced c-Met-mediated MDCKCell Collagen Invasion. FIG. 9A: Modulation of HGF stimulated MDCK cellcollagen I invasion in the Costar transwell chamber invasion assay. 50μl of collagen I was added to the top chamber of the transwell chamber(BD Biosciences) and gelled by adjusting it to a physiological pH with a1:1:1 mixture of 10× Modified Eagle's Medium, 1 M sodium bicarbonate,and 0.1 M sodium hydroxide for 30 minutes at 37° C. 50,000 MDCK cells in1% FBS DMEM were seeded into the inside of the top chamber over thegelled collagen I solution and allowed to attach for two hours at 37° C.After the cells had attached to the collagen I, the indicatedconcentrations of Norleual were added to the top chamber. Additionally,300 μl of 20 ng/ml HGF alone and with 10⁻⁹ M, 10⁻¹⁰ M, and 10⁻¹¹MNorleual was added to the bottom chamber to stimulate invasion throughthe collagen I. The cells were allowed to invade the collagen for twelvehours before they were fixed and stained with Diff-Quik (Dade-Behring).The cells that had invaded the collagen and migrated to the underside ofthe membrane were counted in five random fields at 40× on a lightmicroscope. The effect of treatment was determined using the one-wayANOVA with tukey's post-hoc analysis (Prism statistical analysissoftware). * indicates p<0.01 vs. HGF alone treatment. N=3 pertreatment, mean +/−SEM. FIG. 9B: 50,000 MDCK cells in 1% FBS DMEM wereseeded into the inside of the top chamber and allowed to attach for twohours at 37° C. After the cells had attached, the indicatedconcentrations of Norleual were added to the top chamber. Additionally,300 μl of 20 ng/ml HGF alone and with 10⁻⁹ M, 10⁻¹⁰M, and 10⁻¹¹MNorleual was added to the bottom chamber to stimulate migration throughthe membrane. The cells were allowed to migrate for six hours beforethey were fixed and stained with Diff-Quik (Dade-Behring). The cellsthat had invaded the collagen and migrated to the underside of themembrane were counted in five random fields at 40× on a lightmicroscope. The effect of treatment was determined using the one-wayANOVA with tukey's post-hoc analysis (Prism statistical analysissoftware). * indicates p<0.05 vs. HGF treatment. N=3 per treatment, mean+/−SEM.

FIG. 10 shows Norleual effects on c-Met/HGF stimulated Madin-DarbyCanine Kidney (MDCK) cell proliferation and on MDCK uPA induction. FIG.10A shows attenuation by Norleual of c-Met/HGF stimulated Madin-DarbyCanine Kidney (MDCK) cell proliferation: 50,00 MDCK cells were plated ineach well of a 96 well plate and serum deprived for 24 hours to inducequiescence. Thereafter, 10 ng/ml of HGF alone and with the indicatedconcentrations of Norleual in a 1% FBS DMEM were added to the cells. Thecells were cultured under these conditions for four days. On the fourthday, the treatment media was aspirated and replaced with 5 mg/ml of MTTreagent in 0.1 M glycine buffer (invitrogen). The MTT reagent wasincubated with the cells for four hours at 37° C. before adding DMSO tosolubilize the cells and record the MTT absorbance on a plate reader.The controls were treated as background and subtracted from all of thetreatments to determine the increase in proliferation due solely to HGFtreatment. Differences between the treatments were determined usingone-way ANOVA with Bonferroni's post-hoc analysis (Prism statisticalanalysis software). * indicate p<0.05 vs. HGF treatment. N=6, mean+/−SEM. FIG. 10B shows attenuation by the c-Met antagonist Norleual ofc-Met/HGF urokinase plasminogen activator (uPA) induction. 50,00Madin-Darby Canine Kidney (MDCK) cells were plated in the wells of a 96well plate in 100 μl of complete Dulbecco's Modified Eagle's Medium(DMEM). The cells were serum starved for 24 hours to induce quiescence.Following serum deprivation, 10 ng/ml of Hepatocyte Growth Factor (HGF)alone and with a 10⁻¹⁰ M and 10⁻¹² M Norleual prepared in a 1% FBS DMEMwere added to the cells and the cells were incubated at 37° C. for 48hours under these treatment conditions. After the 48 hour treatment, 10μl of the chromophore labeled tripeptide uPA substrate(Boehringer-Mannheim, Indianapolis, Ind.) was added to the cells andincubated for four hours at 37° C. Absorbance of the cleaved substratewas quantitated on a Biotek Synergy HT 2 plate reader according to themanufacturer's instructions. Controls were considered background andwere subtracted from all treatments. The data was analyzed with Prism®(GraphPad, San Diego, Calif.) statistical analysis software. * indicatesp<0.01 vs. HGF treatment by Tukey's post-hoc analysis. N=6 pertreatment, mean +/−SEM.

FIG. 11 shows Norleual effects in a B16-F10 murine melanoma cellsscratch wound closure assay. FIG. 11A shows Norleual attenuation ofHGF-potentiated scratch closure at 2, 4 and 6 hour timepoints. FIG. 11Bshows relative Norleual effects on Erk1/2 and Akt phosphorylation in theHGF-potentiated B16-F10 murine melanoma cells scratch wound closureassay.

FIG. 12 shows effects of Norleual on murine SA-WAZ-2T breast carcinomacell migration in vitro.

FIG. 13 shows Norleual inhibition of melanoma growth and metastasis invivo. (FIG. 13A) C57BL/6 mice were inoculated subcutaneously withB16-F10 cells. Control or Norleual-containing slow release Elvax pelletswere implanted inter-muscularly. Tumors were measured with digitalcalipers and tumor volumes were calculated (n=6), error bars indicate±SD. (FIG. 13B) 400,000 B16-F10 cells, treated in suspension with eitherNorleual at 10⁻¹¹ M or PBS as a vehicle control, were injected into thetail vein of C57BL/6 mice. Mice received IP injections of Norleual (50μg/kg/day) or PBS vehicle control. After fourteen days, lungs wereremoved and the absorbance of melanin was used to quantify metastasis;bars show (left to right) graft recipients treated with vehicle only,Norelual treated graft recipients, and ungrafted age-matched control.Error bars indicate ±SEM, with n=5 and *p<0.05. (FIG. 13C) Photo ofrepresentative lungs from vehicle-only treated (controls, “Norleal−”)and Norleual treated (“Norleual+”) grafted mice.

FIG. 14 shows Norleual effects on endothelial cell migration in vitro.

FIG. 15 shows the effect of several compounds comprising differentsingle angiotensin-like factors on in vivo angiogenesis as determined byvascularization in a disc assay. Briefly, each compound comprising asingle angiotensin-like factor was placed within a surgical sponge andthe sponge was “sandwiched” in between two impermeable membranes,thereby forming the disc. Next, each disc containing theangiotensin-like factor was implanted subcutaneously in a rat animalmodel and neovascularization was measured after 10 days for eachcompound: Compound 1, (SEQ ID NO:41); Compound 2 (Norleual, SEQ IDNO:43); Compound 3, (SEQ ID NO:47); Compound 4, SEQ ID NO:52[Nle-Tyr-Ile-6(amino) hexanoic acid amide].

FIG. 16 depicts inhibition of angiogenesis by Norleual (Compound 2, SEQID NL:43) in the mouse aortic ring assay. Rings were incubated in EGM-2media (Cambrex) with (n=8) or without (n=6) 100 pM Norleual for fourdays. Digital photos of the rings were taken on day four. FIG. 16A:Quantification of aortic ring angiogenesis. Areas covered by angiogenicsprouts were quantified from day four ring photos, control (−) andNorelual treated (+) (p-value=0.012* and error bars indicate ±SEM). FIG.16B: HUVECs were treated for 5 minutes with HGF and/or Norleual atindicated concentrations. Lysates were IP with anti-c-Met and IB withanti-c-Met or Gab1.

FIG. 17 shows effects on Madin-Darby Canine Kidney (MDCK) Cell Migrationof isolated angiotensin-like factors. Compound 4, SEQ ID NO:52[Nle-Tyr-Ile-6(amino) hexanoic acid amide] was tested for its effect onMDCK cell migration toward an increasing gradient of Hepatocyte GrowthFactor (HGF) in the fluoroblok transwell migration assay (BDBiosciences). MDCK cells were grown to 100% confluency in 100 mm platesand serum deprived for 24 hours to induce quiescence. After 24 hours ofserum deprivation, the cells were fluorescently labeled with 5 μg/ml ofVybrant Dil (Molecular Probes, Eugene, Oreg.) for 30 minutes at 37° C.The cells were washed three times with serum free medium to remove anyresidual dye from the media. The fluorescing cells were detached fromthe plate by adding 3 mls of 0.25% trypsin and incubating for fiveminutes at 37° C. The cells were resuspended in 10 mls of 1% serum mediato neutralize the trypsin and the cells were centrifuged at 1000×g for10 minutes to pellet the cells. The supernatant was aspirated from thecell pellet and the cells were resuspended in 8 mls of fresh 1% FBSDMEM. The cell suspension was triturated to obtain an even cellsuspension. 50,000 cells were seeded in the top chamber of theFluoroblok transwell chamber (BD Biosciences, San Jose, Calif.) and wereallowed to attach for two hours at 37° C. The cells were pre-incubatedwith the peptide (Compound 4) for 15 minutes prior to adding 300 μl of5-10 ng/ml HGF with or without the peptide to the lower chamber tostimulate migration. Compound 4 was used at 10⁻¹⁰ and 10⁻¹¹ molar. Thedata were analyzed with one-way ANOVA followed by Student Newman Keulspost-hoc analysis using NCSS statistical software. P<0.05 for Compound 4vs. HGF as determined by Student Newman Keuls post-hoc analysis.

FIG. 18 shows effects of 6AH derivatives on U87 cell viability. 50,000U87 cells were seeded into each well of a 96 well plate in 10% FBSEagle's Modified Essential Medium (EMEM). After seeding the cells intothe wells, the medium was replaced with a 1% FBS EMEM with or withoutthe indicated peptides. All peptides were tested at 10⁻⁸ M. The cellswere grown for seven days under these treatment conditions. On theseventh day, cell viability was determined by adding 0.2 μM of CalceinAM (Molecular Probes) to the wells and quantitating the amount ofCalcein fluorescence for each well on a fluorescent plate reader. Thetotal number of cells was determined by killing the cells with 100%methanol for 30 minutes and staining the cells with ethidium-1homodimer. Calcein fluorescence is divided by ethidium-1 homodimerfluorescence to determine the live to total cell ratio. 6AH is theparent molecule with Tyrosine in the #2 position. 6AH Asp #2=AsparticAcid in the #2 position, 6AH Trp #2=Tryptophan in the #2 position, and6AH Gly #2=Glycine in the #2 position, 6AH Ser #2=Serine in the #2position, 6AH His=Histidine in the #2 position. The 6AH #1 derivativesinclude Butaric acid, Hexanoic acid, Propionic acid, and Valeric acid.The asterisks indicate p<0.05 versus control by Student Newman Keulspost-hoc analysis.

FIG. 19 depicts effects on angiogenesis of angiotensin-like factors in arat in vivo disc angiogenesis assay. Vascularization was quantified bydigital image analysis of fixed and stained discs recovered from animals14 days after subcutaneous implantation of sponge discs containing theindicated compounds.

DETAILED DESCRIPTION

Certain embodiments of the invention disclosed herein are based on thesurprising discovery that angiotensin-like factors may act as agonistsor antagonists of the c-Met receptor. In particular, and as disclosedherein, it has been discovered that natural and/or synthetic angiotensinpeptides (including angiotensinogen precursor peptides) and otherstructurally related angiotensin-like factors as described herein,including in certain embodiments natural and/or synthetic polypeptidescontaining a consensus amino acid sequence of the general formulaN-aromatic amino acid-hydrophobic amino acid—basic amino acid—C, orhaving another structure as disclosed herein, are capable of altering(i.e. increasing or decreasing in a statistically significant manner,e.g. relative to an appropriate control) a hepatocyte growth factor(HGF) activity or a c-Met receptor activity, for example and accordingto certain non-limiting embodiments, by altering HGF binding to thec-Met receptor.

The presently disclosed compositions and methods will find use in anycontext where it may be desired to alter a hepatocyte growth factoractivity or a c-Met receptor activity, such as in situations whereintervention is sought to alter cell proliferation, migration, adhesion,differentiation or other cellular functions, in processes that mayinclude, for example, one or more of cardiovascular regulation,angiogenesis, inflammation, neuronal growth or regeneration, cognitivedysfunction, adipose tissue deposition, bone regeneration or othertissue repair, or other physiological processes. Embodiments describedherein may thus find therapeutic uses, but the present disclosure is notintended to be so limited and also contemplates diagnostics, screeningassays for identification of therapeutic compounds and/or foridentification of signal transduction pathway components, including suchcomponents that may provide additional or alternative useful therapeutictargets, and other useful applications of the discoveries as presentlydisclosed.

Hepatocyte growth factor (HGF) activities or c-Met receptor activitiesmay in certain preferred embodiments relate to any biological process orevent that results from, contributes to or otherwise involves a specificbinding interaction between (i) HGF (e.g., GenBank Accession No.AAA64239, SEQ ID NO:83) or an angiotensin-like factor that is capable ofspecifically binding to the cell surface c-Met receptor as providedherein, and (ii) the c-Met receptor (e.g., GenBank Accession No.AAA59591, SEQ ID NO:84), that effects a detectable (e.g., withstatistical significance) change (e.g., phosphorylation state or otherstructural and/or functional indicia of an activity state change) in thec-Met receptor and/or in a component of the c-Met receptor signaltransduction pathway.

Hepatocyte growth factor activities or c-Met receptor activities mayinclude induction of cell proliferation, induction of cell scattering,induction of cell migration, induction of specific gene expressionpatterns (which may include both up- and down-regulation of theexpression of one or more particular genes or a coordinated sequence ofdifferential gene expression) or up-regulation or down-regulation ofdownstream intracellular signaling molecules (e.g., changes inphosphorylation states of molecules, such as Gab1). As described herein,induction is intended to refer to either or both of initiating theevent, as well as up-regulating the event (i.e. increasing the event bya statistically significant amount).

One of skill in the art is readily able to measure such activities by,for example in vitro or in vivo assays including radiometricdetermination of cells or cellular components derived therefromfollowing radiolabeling with isotopic molecular precursors (e.g.,analysis of radiolabeled phosphoproteins and/or protein tyrosinephosphate following ³²P labeling; quantification of cellularproliferation by determining incorporated ³H-thymidine; determination ofprotein biosynthesis by incorporation of a radiolabeled amino acid intocellular protein; determination of extracellular matrix (ECM)degradation or biosynthesis by detection of a radiolabeled ECM precursorthat is liberated or incorporated by cells into surrounding ECM, assaysof transcription or transcription factor activity such astranscriptional run-off assays or the like, etc.) and subsequentlymonitoring radioactivity or activity of another detectable indicator;fluorescence microscopy or flow cytometry (e.g., immunocytofluorimetry)of cell populations; qualitative and/or quantitative immunofluorescenceof cells; and using micro-carrier beads to assay cell migration orscattering. (See, for example, Arbab et al., Blood 15: 104 (10) 3410-2(2004); Rosen et al., Exp. Cell Res., 186(1) 22-31 (1990).)

Other hepatocyte growth factor activities or c-Met receptor activitiesmay include cellular extravasation, disruption of extracellular matrix,tumor metastasis, increased angiogenesis, increased tumor size orburden, or other related activities (detectable by, for example, changesin cell adhesion molecule expression and/or by in vivo or in vitromethodologies with which those skilled in the art will be familiar, suchas by histochemistry, immunohistology, emission spectrum scanningmultiphoton microscopy of quantum dot-labeled cells, in vivo opticalimaging, in vivo NMR, functional magnetic resonance imaging, etc.).

Other examples of well known methodologies available for quantifyingcellular proliferation include incorporation of tritiated thymidine intocellular DNA, monitoring of detectable (e.g., fluorimetric orcalorimetric) indicators of cellular respiratory activity, (e.g., MTTassay) or cell counting, or the like. Similarly, in the cell biologyarts there are known multiple techniques for assessing cell survival(e.g., vital dyes, metabolic indicators, etc.) and for determiningapoptosis (e.g., annexin V binding, DNA fragmentation assays, caspaseactivation, PARP cleavage, etc.). Other signaling pathways will beassociated with particular cellular phenotypes, for example specificinduction of gene expression (e.g., detectable as transcription ortranslation products, or by bioassays of such products, or as nuclearlocalization of cytoplasmic factors), altered (e.g., statisticallysignificant increases or decreases) levels of intracellular mediators(e.g., activated kinases or phosphatases, altered levels of cyclicnucleotides or of physiologically active ionic species, etc.), alteredcell cycle profiles, or altered cellular morphology, and the like, suchthat cellular responsiveness to a particular stimulus as provided hereincan be readily identified to determine whether a particular cellcomprises an inducible signaling pathway.

Certain embodiments described herein relate to a biological signalingpathway or signal transduction pathway that comprises a c-Met receptorpathway, which may be induced in subject or biological source cells bycontacting such cells with an appropriate stimulus, which may varydepending upon the signaling pathway under investigation, whether knownor unknown. For example, a signaling pathway that, when induced, resultsin c-Met receptorproten tyrosine phosphorylation and/or c-Met or otherphosphoprotein tyrosine dephosphorylation may be stimulated in subjector biological source cells using any one or more of a variety of wellknown methods and compositions known in the art to stimulate proteintyrosine kinase and/or protein tyrosine phosphatase (PTP) activity.These stimuli may include, without limitation, exposure of cells tocytokines, growth factors, hormones, peptides, small molecule mediators,cell stressors (e.g., ultraviolet light; temperature shifts; osmoticshock; ROS or a source thereof, such as hydrogen peroxide, superoxide,ozone, etc. or any agent that induces or promotes ROS production (see,e.g., Halliwell and Gutteridge, Free Radicals in Biology and Medicine(3^(rd) Ed.) 1999 Oxford University Press, Oxford, UK); heavy metals;alcohol) or other agents that induce c-Met receptor protein tyrosinephosphorylation and/or PTP-mediated phosphoprotein tyrosinedephosphorylation.

As known in the art, for example, HGF under appropriate conditions caninduce a c-Met receptor pathway, such as by altering the phosphorylationstate of the c-Met receptor and/or of a downstream molecular component(e.g., Grb1) of the c-Met receptor pathway. As described herein for thefirst time, isolated AT4 and/or other isolated angiotensin-like factorsas provided herein are capable of specifically binding to the cellsurface c-Met receptor and by so doing can induce one or morealterations in the phosphorylation state of one or more c-Met receptorsignal transduction pathway components.

Other agents that may be capable of inducing a signal transductionpathway may include, for example, interleukins (e.g., IL-1, IL-3),interferons (e.g., IFN-γ), human growth hormone, insulin, epidermalgrowth factor (EGF), platelet derived growth factor (PDGF), granulocytecolony stimulating factor (G-CSF), granulocyte-megakaryocyte colonystimulating factor (GM-CSF), transforming growth factor (e.g., TGF-β1),tumor necrosis factor (e.g., TNF-α) and fibroblast growth factor (FGF;e.g., basic FGF (bFGF)), any agent or combination of agents capable oftriggering T lymphocyte activation via the T cell receptor for antigen(TCR; TCR-inducing agents may include superantigens, specificallyrecognized antigens and/or MHC-derived peptides, MHC peptide tetramers(e.g., Altman et al., 1996 Science 274:94-96) TCR-specific antibodies orfragments or derivatives thereof), lectins (e.g., PHA, PWM, ConA, etc.),mitogens, G-protein coupled receptor agonists such as angiostatin,thrombin, thyrotropin, parathyroid hormone, lysophosphatidic acid (LPA),sphingosine-1-phosphate, serotonin, endothelin, acetylcholine, plateletactivating factor (PAF) or bradykinin, as well as other agents withwhich those having ordinary skill in the art will be familiar (see,e.g., Rhee et al., 10 Oct. 2000 Science's stke,<http:www.stke.org/cgl/content/full/OC_sigtrans;2000/53/pel, andreferences cited therein; see also Gross et al., 1999 J. Biol. Chem.274:26378-86; Prenzel et al., 1999 Nature 402:884-88; Ushio-Fukai etal., 1999 J. Biol. Chem. 274:22699-704; Holland et al., 1998 Endothelium6:113-21; Daub et al., 1997 EMBO J. 16:7032-44; Krypianou et al., 1997Prostate 32:266-71; Marumo et al., 1997 Circulation 96:2361-67).

As noted above, regulated tyrosine phosphorylation contributes tospecific pathways for biological signal transduction, including thoseassociated with cell division, cell survival, apoptosis, proliferationand differentiation, and “inducible signaling pathways” in the contextof the presently disclosed embodiments include transient or stableassociations or interactions among molecular components involved in thecontrol of these and similar processes in cells. Depending on theparticular pathway of interest, an appropriate parameter for determininginduction of such pathway may be selected. For example, for signalingpathways associated with cell proliferation, there is available avariety of well known methodologies for quantifying proliferation,including, for example, incorporation of tritiated thymidine intocellular DNA, monitoring of detectable (e.g., fluorimetric orcolorimetric) indicators of cellular respiratory activity, or cellcounting, or the like. Similarly, in the cell biology arts there areknown multiple techniques for assessing cell survival (e.g., vital dyes,metabolic indicators, etc.) and for determining apoptosis (e.g., annexinV binding, DNA fragmentation assays, caspase activation, etc.). Othersignaling pathways will be associated with particular cellularphenotypes, for example specific induction of gene expression (e.g.,detectable as transcription or translation products, or by bioassays ofsuch products, or as nuclear localization of cytoplasmic factors),altered (e.g., statistically significant increases or decreases) levelsof intracellular mediators (e.g., activated kinases or phosphatases,altered levels of cyclic nucleotides or of physiologically active ionicspecies, etc.), or altered cellular morphology, and the like, such thatcellular responsiveness to a particular stimulus as provided herein canbe readily identified to determine whether a particular cell comprisesan inducible signaling pathway.

A number of methods are described herein and known in the art fordetection of one or more particular signal transduction pathwaycomponent polypeptides such as c-Met receptor pathway components, andfor determination of whether such polypeptides may betyrosine-phosphorylated in cells following stimulation as describedherein. Also described herein are methods for detecting suchpolypeptides, including determination of altered (i.e., increased ordecreased with statistical significance) tyrosine phosphorylation thatmay further include determination of the phosphorylation state ofparticular tyrosine residues at specified positions within a polypeptidesequence, which altered tyrosine phosphorylation may in certainembodiments be accompanied by the presence or absence of induction ofone or more HGF activities or c-Met receptor activities in the cellsfrom which such polypeptides are obtained (e.g., as a result of exposureto a stimulus, such as HGF and/or an angiotensin-like factor as providedherein).

Non-limiting examples of such detection methods include the use ofreagents that specifically bind to c-Met receptor signaling pathwaycomponents, for example, by immunological methods (e.g.,immunoprecipitation, immunoblotting, ELISA, radioimmunoprecipitation,and the like) that employ antibodies as provided herein that are capableof specifically binding a particular signaling pathway componentpolypeptide or a particular tyrosine-phosphorylated polypeptide.

Additionally and as described in greater detail herein, in certainembodiments induction of one or more HGF activities or c-Met receptoractivities induced by a stimulus may be partially or completelyimpaired, abrogated, inhibited or otherwise counteracted by inclusion ofan angiotensin-like factor that is capable of specifically binding to acell surface c-Met receptor, for instance, by virtue of being able toinhibit competitively the binding to the cell surface c-Met receptor ofa native HGF hinge region polypeptide having the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82].

The surprising discovery disclosed herein, that the c-Met receptor actsas a functional biological receptor for AT(4) and other AT(4) receptorligands, including angiotensin-like factors as provided herein, permitsadvantageously contacting a cell or plurality of cells with such anisolated angiotensin-like factor that is capable of specifically bindingto a cell surface c-Met receptor, under conditions and for a timesufficient for the angiotensin-like factor to interact with the cellsurface c-Met receptor, to provide a method for altering a hepatocytegrowth factor activity or a c-Met receptor activity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring nucleic acid orpolypeptide present in a living animal is not isolated, but the samenucleic acid or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Such nucleicacid could be part of a vector and/or such nucleic acid or polypeptidecould be part of a composition (e.g., a cell lysate), and still beisolated in that such vector or composition is not part of the naturalenvironment for the nucleic acid or polypeptide. The term “gene” meansthe segment of DNA involved in producing a polypeptide chain; itincludes regions preceding and following the coding region “leader andtrailer” as well as intervening sequences (introns) between individualcoding segments (exons).

In particular, according to certain preferred embodimentsangiotensin-like factors may comprise peptides, polypeptides orpeptidomimetics that include, or that share close sequence identity toor structural features with, amino acids 122-128 of the hepatocytegrowth factor precursor polypeptide having the amino acid sequence setforth in SEQ ID NO:2 (in which the amino acids at sequence positions122-128 are Lys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82], also referred toas the HGF “hinge region”) or the closely related sequence set forth inSEQ ID NO:1 (Leu-Asp-Tyr-Ile-Arg-Asn-Cys). Polypeptides of 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids havingsuch a HGF hinge region polypeptide structure including polypeptideshaving at least 75%, 80%, 85%, 90% or 95% sequence identity or sequencehomology thereto, or peptidomimetics thereof, have been found to beespecially effective in out-competing the native hepatocyte growthfactor for binding to the c-Met receptor.

As generally referred to in the art, and as used herein, sequenceidentity and sequence homology may be used interchangeably and generallyrefer to the percentage of nucleotides or amino acid residues in acandidate sequence that are identical with, respectively, thenucleotides or amino acid residues in a native polynucleotide orpolypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Preferably, an angiotensin-like factor polypeptide or encodingpolynucleotide of the embodiments disclosed herein shares at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, 96%, 97%, 98%, or 99% of the amino acid residues (or ofthe nucleotides in a polynucleotide encoding such an antiotensin-likefactor polypeptide) with the HGF hinge region target sequence. Suchsequence identity may be determined according to well known sequenceanalysis algorithms, including those available from the University ofWisconsin Genetics Computer Group (Madison, Wis.), such as FASTA, Gap,Bestfit, BLAST, or others.

It has also been determined according to certain embodiments of thepresent invention that N-terminus extensions of the angiotensin-likefactors can alter the affinity of the angiotensin-like factor binding tothe c-Met receptor, while C-terminus extensions can enhance bindingand/or activity of the angiotensin-like factor. Another advantage of thecompositions of certain herein disclosed embodiments is that most areeffective at very low doses (for example, in the nanomolar to picomolarrange). Furthermore, among the herein described angiotensin-like factorsmay be found c-Met receptor antagonists that are generally capable offunctioning as anti-angiogenic as well as anti-tumor agents.

According to certain presently disclosed embodiments, anangiotensin-like factor comprises a polypeptide of no more than 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids,or a peptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I]

wherein N is an amino terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, C is a carboxy terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, X₁ is phenylalanine, tryptophan ortyrosine, X₂ is isoleucine, leucine, alanine, valine, phenylalanine,proline, methionine or tryptophan, and X₃ is lysine, arginine orhistidine.

“Natural or non-natural amino acid” includes any of the common naturallyoccurring amino acids which serve as building blocks for thebiosynthesis of peptides, polypeptides and proteins (e.g., alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine)and also includes modified, derivatized, enantiomeric, rare and/orunusual amino acids, whether naturally occurring or synthetic, forinstance, hydroxyproline, hydroxylysine, desmosine, isodesmosine,ε-N-methyllysine, ε-N-trimethyllysine, methylhistidine, dehydrobutyrine,dehydroalanine, α-aminobutyric acid, β-alanine, γ-aminobutyric acid,homocysteine, homoserine, citrulline, ornithine and other amino acidsthat may be isolated from a natural source and/or that may be chemicallysynthesized, for instance, as may be found in Proteins, Peptides andAmino Acids Sourcebook (White, J. S, and White, D.C., 2002 Humana Press,Totowa, N.J.) or in Amino Acid and Peptide Synthesis (Jones, J., 2002Oxford Univ. Press USA, New York) or in Unnatural Amino Acids, ChemFilesVol. 1, No. 5 (2001 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, Mo.) orin Unnatural Amino Acids II, ChemFiles Vol. 2, No. 4 (2002 Fluka ChemieGmbH; Sigma-Aldrich, St. Louis, Mo.). Additional descriptions of naturaland/or non-natural amino acids may be found, for example, in Kotha, 2003Acc. Chem. Res. 36:342; Maruoka et al., 2004 Proc. Nat. Acad. Sci. USA101:5824; Lundquist et al., 2001 Org. Lett. 3:781; Tang et al., 2002 J.Org. Chem. 67:7819; Rothman et al., 2003 J. Org. Chem. 68:6795; Krebs etal., 2004 Chemistry 10:544; Goodman et al., 2001 Biopolymers 60:229;Sabat et al., 2000 Org. Lett. 2:1089; Fu et al., 2001 J. Org. Chem.66:7118; and Hruby et al., 1994 Meths. Mol. Biol. 35:249. The standardthree-lefter abbreviations and 1-letter symbols are used herein todesignate natural and non-natural amino acids.

Other non-natural amino acids or amino acid analogues are known in theart and include, but are not limited to, non-natural L or D derivatives,fluorescent labeled amino acids, as well as specific examples includingO-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, 3-idio-tyrosine, O-propargyl-tyrosine,homoglutamine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a3-nitro-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, afluorinated phenylalanine, an isopropyl-L-phenylalanine, ap-azido-L-phenylalanine, a p-acyl-L-phenylalanine, ap-acetyl-L-phenylalanine, an m-acetyl-L-phenylalanine, selenomethionine,telluromethionine, selenocysteine, an alkyne phenylalanine, anO-allyl-L-tyrosine, an O-(2-propynyl)-L-tyrosine, ap-ethylthiocarbonyl-L-phenylalanine, ap-(3-oxobutanoyl)-L-phenylalanine, a p-benzoyl-L-phenylalanine, anL-phosphoserine, a phosphonoserine, a phosphonotyrosine,homoproparglyglycine, azidohomoalanine, a p-iodo-phenylalanine, ap-bromo-L-phenylalanine, dihydroxy-phenylalanine,dihydroxyl-L-phenylalanine, a p-nitro-L-phenylalanine, anm-methoxy-L-phenylalanine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, trifluoroleucine, norleucine,5-fluoro-tryptophan, para-halo-phenylalanine, seleno-methionine,ethionine, S-nitroso-homocysteine, thia-proline, 3-thienyl-alanine,homo-allyl-glycine, trifluoroisoleucine, trans andcis-2-amino-4-hexenoic acid, 2-butynyl-glycine, allyl-glycine,para-azido-phenylalanine, para-cyano-phenylalanine,para-ethynyl-phenylalanine, hexafluoroleucine, 1,2,4-triazole-3-alanine,2-fluoro-histidine, L-methyl histidine, 3-methyl-L-histidine,β-2-thienyl-L-alanine, β-(2-thiazolyl)-DL-alanine, homoproparglyglycine(HPG) and azidohomoalanine (AHA) and the like.

In certain embodiments a natural or non-natural amino acid may bepresent that comprises an aromatic side chain, as found, for example, inphenylalanine or tryptophan or analogues thereof including in othernatural or non-natural amino acids based on the structures of which theskilled person will readily recognize when an aromatic ring system ispresent, typically in the form of an aromatic monocyclic or multicyclichydrocarbon ring system consisting only of hydrogen and carbon andcontaining from 6 to 19 carbon atoms, where the ring system may bepartially or fully saturated, and which may be present as a group thatincludes, but need not be limited to, groups such as fluorenyl, phenyland naphthyl.

In certain embodiments a natural or non-natural amino acid may bepresent that comprises a hydrophobic side chain as found, for example,in alanine, valine, isoleucine, leucine, proline, phenylalanine,tryptophan or methionine or analogues thereof including in other naturalor non-natural amino acids based on the structures of which the skilledperson will readily recognize when a hydrophobic side chain (e.g.,typically one that is non-polar when in a physiological milieu) ispresent. In certain embodiments a natural or non-natural amino acid maybe present that comprises a basic side chain as found, for example, inlysine, arginine or histidine or analogues thereof including in othernatural or non-natural amino acids based on the structures of which theskilled person will readily recognize when a basic (e.g., typicallypolar and having a positive charge when in a physiological milieu) ispresent.

Angiotensin-like factor polypeptides and proteins disclosed herein mayinclude L- and/or D-amino acids so long as the biological activity ofthe polypeptide is maintained. The isolated angiotensin-like factorpolypeptides and proteins also may comprise in certain embodiments anyof a variety of known natural and artificial post-translational orpost-synthetic covalent chemical modifications by reactions that mayinclude glycosylation (e.g., N-linked oligosaccharide addition atasparagine residues, O-linked oligosaccharide addition at serine orthreonine residued, glycation, or the like), fatty acylation,acetylation, PEGylation, and phosphorylation. Polypeptides hereindisclosed may further include analogs, alleles and allelic variantswhich may contain amino acid deletions, or additions or substitutions ofone or more amino acid residues with other naturally occurring aminoacid residues or non-natural amino acid residues.

Peptide and non-peptide analogs may be referred to as peptide mimeticsor peptidomimetics, and are known in the pharmaceutical industry(Fauchere, J. Adv. Drug Res. 15:29 (1986); Evans et al. J. Med. Chem.30: 1229 (1987)). These compounds may contain one or more non-naturalamino acid residue(s), one or more chemical modification moieties (forexample, glycosylation, pegylation, fluorescence, radioactivity, orother moiety), and/or one or more non-natural peptide bond(s) (forexample, a reduced peptide bond: —CH₂—NH₂—). Peptidomimetics may bedeveloped by a variety of methods, including by computerized molecularmodeling, random or site-directed mutagenesis, PCR-based strategies,chemical mutagenesis, and others.

Hence according to certain presently disclosed embodiments anangiotensin-like factor may comprise a polypeptide of no more than 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I]

wherein N is an amino terminus of the peptide or peptidomimetic andconsists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or17 amino acids that are independently selected from natural andnon-natural amino acids, C is a carboxy terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, X₁ is a natural or non-naturalamino acid having an aromatic side chain, X₂ is a natural or non-naturalamino acid having a hydrophobic side chain, and X₃ is a natural ornon-natural amino acid having a basic side chain.

Accordingly in these and other embodiments it will be appreciated thatthe amino terminus of certain angiotensin-like factors may consist of1-17 independently selected natural or non-natural amino acids, and/orthat in certain embodiments the carboxy terminus of the angiotensin-likefactor may consist of 1-17 independently selected natural or non-naturalamino acids, where such amino and carboxy termini may have any sequenceso long as the isolated angiotensin-like factor is of no more than 3-20amino acids and comprises N—X₁—X₂—X₃—C as recited herein, and is capableof specifically binding to the cell surface c-Met receptor.

Disclosed herein are a number of representative angiotensin-like factorsthat comprise N—X₁—X₂—X₃—C as recited herein and that are capable ofspecifically binding to the cell surface c-Met receptor, such that inview of the present disclosure those familiar with the art will be ablereadily to make and use additional angiotensin-like factors according tothe present embodiments. For example, determination of thethree-dimensional structures of representative angiotensin-like factorsmay be made through routine methodologies such that substitution of oneor more amino acids with selected natural or non-natural amino acids canbe virtually modeled for purposes of determining whether a so derivedstructural variant retains the space-filling properties of disclosedspecies. See, for example, Bradley et al., Science 309: 1868-1871(2005); Schueler-Furman et al., Science 310:638 (2005). Some additionalnon-limiting examples of computer algorithms that may be used for theseand related embodiments, such as for rational design of angiotensin-likefactors as provided herein, include Desktop Molecular Modeler (See, forexample, Agboh et al., J. Biol. Chem., 279, 40: 41650-57 (2004)), whichallows for determining atomic dimensions from spacefilling models (vander Waals radii) of energy-minimized conformations; GRID, which seeks todetermine regions of high affinity for different chemical groups,thereby enhancing binding, Monte Carlo searches, which calculatemathematical alignment, and CHARMM (Brooks et al. (1983) J. Comput.Chem. 4:187-217) and AMBER (Weiner et al (1981) J. Comput. Chem. 106:765), which assess force field calculations, and analysis (see also,Eisenfield et al. (1991) Am. J. Physiol. 261:C376-386; Lybrand (1991) J.Pharm. Belg. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbamet al. (1990) Proteins 7:99-111; Pedersen (1985) Environ. HealthPerspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn.9:475-488).

In certain other presently disclosed embodiments the isolatedangiotensin-like factor may comprise a polypeptide of no more than 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, said polypeptide comprising (i) atripeptide having an amino acid sequence that is selected from the groupconsisting of: Lys-Asp-Tyr, Leu-Asp-Tyr, Asp-Tyr-Ile, Tyr-Ile-Arg,Ile-Arg-Asn, and Arg-Asn-Cys, and (ii) at least one of an amino terminusand a carboxy terminus as described herein, each of said amino terminusand said carboxy terminus consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, as also describedherein. In certain related embodiments such a polypeptide orpeptidomimetic comprises the amino acid sequenceNle-Tyr-Leu-Ψ-His-Pro-Phe as set forth in SEQ ID NO:43, wherein Ψconsists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II]

In certain other presently disclosed embodiments the isolatedangiotensin-like factor may comprise the polypeptide or peptidomimeticof general formula [I] as described above and which comprises at leastone amino acid sequence selected from the group consisting of SEQ IDNOS:9, 25, 33, 41, 42, 45-50, 52-80 and 81, which sequences are setforth in the Sequence Listing and are also presented here: (SEQ ID NO:9)Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, (SEQ ID NO:25)Arg-Val-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:33) Val-Tyr-Ile-His-Pro-Phe,(SEQ ID NO:41) Nle-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:42)Val-ψ-Tyr-Leu-ψ-His-Pro-Phe, (SEQ ID NO:45) Nle-ψ-Tyr-Ile-His-Pro-Phe,(SEQ ID NO:46) Leu-ψ-Tyr-Leu-ψ-His-Pro-Phe, (SEQ ID NO:47)Nle-Tyr-Ile-His, (SEQ ID NO:48) Nle-Tyr-Ile-(CH₂)₆-Phe-amide, (SEQ IDNO:49) Nle-Tyr-Ile-Sar-Sar-dPhe, (SEQ ID NO:Asp-Arg-Val-Tyr-Ile-HIs-Pro-Phe-His-Leu-Leu-Val- Tyr, [SEQ ID NO:52]Nle-Tyr-Ile-6(amino) hexanoic acid amide, (SEQ ID NO:53)Nle-Tyr-Ile-His-Pro, (SEQ ID NO:54) Lys-Tyr-Ile-His-Pro-Phe, (SEQ IDNO:55) benzyl-cysteine-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:56)dNle-Tyr-Ile-His-Pro-Phe, [SEQ ID NO:57] Nle-Tyr-Ile-ψ-His-Pro-Phe, [SEQID NO:58] Nle-Tyr-Val-ψ-His-Pro-Phe, [SEQ ID NO:59]Nle-Tyr-Nle-ψ-His-Pro-Phe, [SEQ ID NO:60] Nle-Phe-Leu-ψ-His-Pro-Phe,[SEQ ID NO:61] Nle-Phe-Ile-ψ-His-Pro-Phe, [SEQ ID NO:62]Nle-Phe-Val-ψ-His-Pro-Phe, [SEQ ID NO:63] Nle-Phe-Nle-ψ-His-Pro-Phe,[SEQ ID NO:64] Nle-Tyr-Leu-ψ-Arg-Pro-Phe, [SEQ ID NO:65]Nle-Tyr-Ile-ψ-Arg-Pro-Phe, [SEQ ID NO:66] Nle-Tyr-Val-ψ-Arg-Pro-Phe,[SEQ ID NO:67] Nle-Tyr-Nle-ψ-Arg-Pro-Phe, [SEQ ID NO:68]Nle-Phe-Leu-ψ-Arg-Pro-Phe, [SEQ ID NO:69] Nle-Phe-Ile-ψ-Arg-Pro-Phe,[SEQ ID NO:70] Nle-Phe-Val-ψ-Arg-Pro-Phe, [SEQ ID NO:71]Nle-Phe-Nle-ψ-Arg-Pro-Phe, [SEQ ID NO:72] Nle-Tyr-Leu-ψ-Lys-Pro-Phe,[SEQ ID NO:73] Nle-Tyr-Ile-ψ-Lys-Pro-Phe, [SEQ ID NO:74]Nle-Tyr-Val-ψ-Lys-Pro-Phe, [SEQ ID NO:75] Nle-Tyr-Nle-ψ-Lys-Pro-Phe,[SEQ ID NO:76] Nle-Phe-Leu-ψ-Lys-Pro-Phe, [SEQ ID NO:77]Nle-Phe-Ile-ψ-Lys-Pro-Phe, [SEQ ID NO:78] Nle-Phe-Val-ψ-Lys-Pro-Phe,[SEQ ID NO:79] Nle-Phe-Nle-ψ-Lys-Pro-Phe, [SEQ ID NO:80] γ-aminobutyricacid-Tyr-Ile, and [SEQ ID NO:81] β-Ala-Tyr-Ile,

wherein Ψ consists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II].

Examples of angiotensin-like factors that may be used according tocertain embodiments include peptides, polypeptides or peptidomimeticsthereof having the following structural formulae, where it is to beunderstood that according to certain other embodiments it is expresslycontemplated that one or more of the following peptides orpeptidomimetics thereof may not be used. Accordingly, in these andrelated embodiments an angiotensin-like factor may include at least onepeptide or polypeptide, or a peptidomimetic thereof, comprising any oneof: at least amino acids 4-6 of the native angiotensinogen precursor(SEQ ID NO:50), at least amino acids 4-6 of native angiotensin I (SEQ IDNO:9), at least amino acids 4-6 of native angiotensin II (SEQ ID NO:17),at least amino acids 4-6 of native angiotensin III (SEQ ID NO:25),native angiotensin IV (SEQ ID NO:33); COMPOUND 1 (SEQ ID NO:41)[Nle-Tyr-Ile-His-Pro-Phe]; COMPOUND 2 (SEQ ID NO:43)[Nle-Tyr-Leu-Ψ-His-Pro-Phe]; COMPOUND 3 (SEQ ID NO:47)[Nle-Tyr-Ile-His]; COMPOUND 4 [Nle-Tyr-Ile-6(amino) hexanoic acid amide](SEQ ID NO:52); COMPOUND 5 (SEQ ID NO:42) [Val-Ψ-Tyr-Leu-Ψ-His-Pro-Phe];COMPOUND 6 (SEQ ID NO:45) [Nle-Ψ-Tyr-Ile-His-Pro-Phe]; COMPOUND 8 (SEQID NO:46) [Leu-Ψ-Tyr-Leu-Ψ-His-Pro-Phe]; COMPOUND 9 (SEQ ID NO:48)[Nle-Tyr-Ile-(CH₂)₆-Phe-amide]; COMPOUND 10 (SEQ ID NO:49)[Nle-Tyr-Ile-Sar-Sar-dPhe], COMPOUND 11 (SEQ ID NO:53)[Nle-Tyr-Ile-His-Pro]; COMPOUND 12 (SEQ ID NO:54)[Lys-Tyr-Ile-His-Pro-Phe]; COMPOUND 13 (SEQ ID NO:55)[benzyl-cysteine-Tyr-Ile-His-Pro-Phe]; COMPOUND 14 [γ-aminobutyricacid-Tyr-Ile]; COMPOUND 15 [β-Ala-Tyr-Ile]; COMPOUND 16 (SEQ ID NO:56)[dNle-Tyr-Ile-His-Pro-Phe]; Nle-Tyr-Ile-Ψ(CH2-NH2)-His-Pro-Phe [SEQ IDNO:57], Nle-Tyr-Val-Ψ(CH2-NH2)-His-Pro-Phe [SEQ ID NO:58],Nle-Tyr-Nle-Ψ(CH2-NH2)-His-Pro-Phe [SEQ ID NO:59],Nle-Phe-Leu-Ψ(CH2-NH2)-His-Pro-Phe [SEQ ID NO:60],Nle-Phe-Ile-Ψ(CH2-NH2)-His-Pro-Phe [SEQ ID NO:61],Nle-Phe-Val-Ψ(CH2-NH2)-His-Pro-Phe [SEQ ID NO:62],Nle-Phe-Nle-Ψ(CH2-NH2)-His-Pro-Phe [SEQ ID NO:63],Nle-Tyr-Leu-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:64],Nle-Tyr-Ile-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:65],Nle-Tyr-Val-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:66],Nle-Tyr-Nle-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:67],Nle-Phe-Leu-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:68],Nle-Phe-Ile-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:69],Nle-Phe-Val-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:70],Nle-Phe-Nle-Ψ(CH2-NH2)-Arg-Pro-Phe [SEQ ID NO:71],Nle-Tyr-Leu-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:72],Nle-Tyr-Ile-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:73],Nle-Tyr-Val-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:74],Nle-Tyr-Nle-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:75],Nle-Phe-Leu-(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:76],Nle-Phe-Ile-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:77],Nle-Phe-Val-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:78], andNle-Phe-Nle-Ψ(CH2-NH2)-Lys-Pro-Phe [SEQ ID NO:79], wherein Ψ consists ofa reduced peptide bond of formula II:—CH₂—NH₂—  [II].

Certain other herein disclosed embodiments relate to an isolatedangiotensin-like factor that comprises an antibody, including antibodiesthat specifically bind to the c-Met receptor, and further includingthose that compete successfully with HGF for binding to the c-Metreceptor. Antibodies in these and related embodiments still further mayinclude, in preferred embodiments, antibodies that structurally and/orfunctionally mimic the HGF hinge region (Lys-Asp-Tyr-Ile-Arg-Asn-Cys,[SEQ ID NO:82]). Accordingly and in certain related embodiments, it iscontemplated for the first time that antibodies may be selected fortheir ability specifically to inhibit competitively the bindinginteraction between an HGF hinge region as described herein (e.g., apolypeptide that comprises SEQ ID NO:82) and a c-Met receptor. Suchantibodies may also in certain embodiments include an anti-idiotypeantibody that may be generated against, and specific for, one or morecomplementarity determining region (CDR) of an immunoglobulin thatspecifically binds to a HGF hinge region epitope, for example thatspecifically binds to all or a portion of SEQ ID NO:82 as may occur in anative HGF polypeptide, thereby to find use of such an anti-idiotypeantibody as an angiotensin-like factor as provided herein.

Antibodies may be obtained from a variety of sources and may compriseone or more immunoglobulins of any class or subclass (for example, IgG,IgM, IgD, IgE, IgA, or any combination thereof, including, e.g., humanIgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂, etc., murine IgG₁, IgG_(2A),IgG_(2B), etc.) (Harlow, E., and Lane, D., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 343-348(1988)). Antibody-producing cells may undergo isotype class switchingsuch that a nucleic acid molecule encoding an Ig V_(L) or V_(H) isisolated using methods known in the art and such that it does notinclude any nucleic acid sequences encoding C_(L) or C_(H). The nucleicacid molecule encoding V_(L) or V_(H) is then operatively linked to anucleic acid sequence encoding a C_(L) or C_(H) from a different classof immunoglobulin molecule. This may be accomplished by using a vectoror nucleic acid molecule that comprises a polynucleotide coding sequencefor the C_(L) or C_(H) chain, thus switching the antibody from one classto another. The antibody may then be expressed in a cell and collectedtherefrom to obtain the antibody of the desired isotype.

Antibodies according to these and related embodiments, for use asangiotensin-like factors, may encompass intact immunoglobulin moleculesas well as anti-idiotypic antibodies, mutants, fusion proteins,monoclonal antibodies (including chimeric antibodies, “humanized”antibodies, “primatized” antibodies, etc.), polyclonal antibodies,multi-specific antibodies (including bispecific antibodies), affinitymatured, as well as antibody fragments of any of these (including Fv,Fab, Fab′, F(ab′)₂, etc.), heteroconjugate antibodies, immunoconjugates,immunoliposomes, or other modifications that comprise an immunoglobulinantigen recognition site of an appropriate specificity, such as anantigen binding site that is capable of specifically binding to a cellsurface c-Met receptor in a manner that competitively inhibits HGFbinding via the HGF hinge region (SEQ ID NO:82) to the c-Met receptor.Non-limiting examples of forms of antibodies that may be used in theseand related embodiments include diabodies, minibodies, Janusins and thelike. Antibodies may be produced by any means known in the art,including recombinant or chemical synthesis, or purification fromhybridoma cell lines.

In addition, antibodies may be labeled or marked by chemical, physicalor physico-chemical means including by conjugation to a detectable agentfor use in diagnostic or therapeutic uses. Some examples of detectableagents include enzymes, radioisotopes, fluorescent compounds, colloidalmetals, chemiluminescent compounds, and bioluminescent compounds. Oneskilled in the art will know how readily to implement these or othersuitable detectable agents according to relevant immunochemicalmethodologies by no more than routine experimentation. Antibodies mayalso, according to related embodiments, be covalently or non-covalentlybound to any number of carriers including solid-phase carriers, whetheractive or inert, and including glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agarose, magnetite and other suitable carriers.

Certain embodiments contemplate angiotensin-like factors that may beprovided as humanized antibodies, which may be obtained, for example, bysubstituting C_(H)1, C_(H)2, C_(H)3, immunoglobulin hinge domains and/orimmunoglobulin framework domains of a non-human antibody with thecorresponding human sequence(s) while maintaining one or more or all ofthe CDRs of the heavy chain or light chain or both. Chimeric antibodiesare thus contemplated and generally include antibodies in which aportion of the heavy and/or light chain is identical with or homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies. (See U.S. Pat. No. 4,816,567.)

Certain embodiments of the present invention include substantially pureor isolated antibodies, which refers to antibodies that have beenremoved from native or naturally occurring antibodies and the purifiedor isolated antibodies are substantially free of contaminants relatingto a native or naturally occurring antibody environment.

In other embodiments, there are provided nucleic acid molecules, vectorsand host cells that may be used to make mutated antibodies. Suchantibodies may be mutated in the variable domains of the heavy and/orlight chains to alter a binding property of the antibody (for example,to change the binding affinity), or may be made in a framework region orconstant domain (for example, to increase the half-life of theantibody). In still other embodiments, a fusion antibody orimmunoadhesin may be made which comprises all or a portion of theantibody linked to another polypeptide. For example, the variable regionmay be linked to another polypeptide, or the V_(H) domain may be linkedto a first polypeptide while the V_(L) domain is linked to a secondpolypeptide that interacts with the first polypeptide. The resultingfusion antibodies may be used to target a particular tissue or cell, asthe polypeptide(s) may contain therapeutic agents, such as a toxin,growth factor, or other regulatory protein, or may be a diagnosticagent, such as an enzyme that can be visualized or tracked. Furthermore,fusion antibodies may be generated with two or more single-chainantibodies linked together, forming a divalent or polyvalent antibody ona single polypeptide chain, or a bispecific antibody.

In other embodiments, single chain Fv or scFV antibodies are alsocontemplated For example, Kappa bodies (Ill et al., Prot. Eng. 10:949-57 (1997); minibodies (Martin et al., EMBO J. 13: 5305-9 (1994);diabodies (Holliger et al., PNAS 90: 6444-8 (1993); or Janusins(Traunecker et al., EMBO J. 10: 3655-59 (1991) and Traunecker et al.Int. J. Cancer Suppl. 7: 51-52 (1992), may be prepared using standardmolecular biology techniques following the teachings of the presentapplication with regard to selecting antibodies having the desiredspecificity. In still other embodiments, bispecific or chimericantibodies may be made that encompass the ligands of the presentinvention. For example, a chimeric antibody may comprise CDRs andframework regions from different antibodies, while bispecific antibodiesmay be generated that bind specifically to c-Met through one bindingdomain and to a second molecule through a second binding domain. Theseantibodies may be produced through recombinant molecular biologicaltechniques or may be physically conjugated together.

Any of the antibodies considered herein may be derivatized or linked toanother molecule (such as a peptide or other label). Typically, theantibodies or portions thereof (whether intact or modified in form) maybe derivatized such that the c-Met binding is not adversely affected.For instance, an antibody or antibody portion of the presently disclosedembodiments may be linked (by chemical coupling, genetic fusion,noncovalent association or otherwise), to one or more other agentsincluding another antibody, detection agent, cytotoxic agent,therapeutic agent, and/or a protein or peptide.

Any angiotensin-like factor as provided herein that is capable ofaltering (e.g., increasing or decreasing in a statistically significantmanner) a hepatocyte growth factor activity or a c-Met receptor activityand that is capable of specifically binding to a cell surface c-Metreceptor may be used to practice certain presently disclosedembodiments, including a native sequence angiotensin molecule or aprecursor, fragment or functional subunit thereof, or a peptidehomologue or analogue thereof including an organic small molecule orpeptide, and/or a polypeptide variant of a native sequence c-Metreceptor ligand.

It should be noted that according to certain preferred embodimentsdescribed herein, an angiotensin-like factor comprises all or a portionof an isolated naturally or non-naturally occurring molecule that iscapable of functionally and/or structurally mimicking an HGF hingeregion, for example, SEQ ID NO: 82 or a polypeptide having 5, 6 or 7 ofthe amino acids that are present at positions 122-128 of SEQ ID NO:1, ora peptide or non-peptide analogue thereof as described herein and aswill be appreciated by one skilled in the art based on the presentdisclosure.

Certain related embodiments contemplate a functional molecule, for useas an angiotensin-like factor, that may be capable of producing the sameor similar biological effects (i.e., alteration of a HGF activity or ac-Met receptor activity) as those which result from interaction of aspecific herein disclosed angiotensin-like factor with a cell surfacec-Met receptor, which functional molecule may occur as an organic smallmolecule, a peptide, an antibody, a glycopeptide, a glycolipid, apolysaccharide, an oligosaccharide, a nucleic acid, a peptidomimetic, ananti-sense polynucleotide, a ribozyme, a nucleic acid triple helix, or aderivative, metabolite, catabolite, precursor, prodrug, or analogthereof, including isolated enantiomeric, diastereomeric and geometricisomers thereof, and mixtures thereof; or a composition or medicamentthat includes said compound or mixture comprising compounds as describedherein.

Thus, an angiotensin-like factor according to certain herein disclosedembodiments may refer to part of any angiotensin molecule, including theangiotesinogen precursor molecule, or any part of an angiotensinmolecule for which no known receptor has yet been identified. Sincepresently known or isolated angiotensin peptides are related spliceproducts of the angiotensinogen precursor (see, e.g., FIG. 1), eachangiotensin peptide shares amino acid sequence identity, in particularin the regions toward the amino terminal end and middle sections of eachangiotensin peptide. As described herein for certain embodiments, theseregions appear to be relevant for binding of angiotensin-like factors tothe c-Met receptor. Thus, certain embodiments, such as the methods ofdiagnosing, preventing or treating a condition associated with c-Metreceptor dysregulation, relate to the use of an angiotensin-like factorthat may comprise the amino acid (or encoding nucleic acid) sequencesthat are common among angiotensin peptides, including theangiotensinogen precursor, angiotensin I, angiotensin II, angiotensinIII, and angiotensin IV. In certain such embodiments, theangiotensin-like factor may therefore, in addition to being capable ofbinding to a cell surface c-Met receptor, also be capable of binding toone or several other angiotensin receptors, which in some embodimentsmay include an angiotensin II receptor and in certain other embodimentsmay include an angiotensin IV receptor (e.g., IRAP).

However, an angiotensin-like factor according to certain embodimentsdisclosed herein expressly may not encompass the known full-lengthnative sequence of the angiotensinogen precursor, or any of the knownfull-length native sequences of the angiotensin I, II, III, or IVpeptides or nucleic acids.

Certain embodiments relate to nucleic acid molecules encoding anangiotensin-like factor. Methods for production of desired nucleic acidsand/or polypeptides are well known in the art. For example, nucleicacids and/or polypeptides may be isolated from cells or synthesized denovo by chemical synthesis. Such nucleic acids or polypeptides may beincorporated into a vector, and transformed into a host cell. Host cellsmay be cultured in standard nutrient media plus necessary supplements oradditives for inducing promoters, selecting transformants or amplifyingthe appropriate sequences.

As also described above, certain embodiments also relate topeptidomimetics, or “artificial” polypeptides. Such polypeptides maycontain one or more amino acid insertions, deletions or substitutions,one or more altered or artificial peptide bond, one or more chemicalmoiety (such as polyethylene glycol, glycosylation, label, toxin, orother moiety), and/or one or more non-natural amino acid. Synthesis ofpeptidomimetics is well known in the art and may include alteringnaturally occurring proteins or polypeptides by chemical mutagenesis,single or multi-site-directed mutagenesis, PCR shuffling, use of alteredaminoacyl tRNA or aminoacyl tRNA synthetase molecules, the use of “stop”codons such as amber suppressors, use of four or five base-pair codons,or other means.

Without wishing to be bound by any particular theory, tumor angiogenesismay be promoted by HGF secretion from vascular endothelial cells. Oncethe endothelial cells are stimulated, proteinases are activated thatdegrade the local extracellular matrix, which allows the cells to moveinto the stroma. The endothelial cells proliferate and may ultimatelyform tubular structures that are capable of fusing with other bloodvessels. Tumor angiogenesis is typically not as well orchestrated asangiogenesis related to embryogenesis, corpus luteum formation, woundhealing (including dermal, epidermal or other skin wounds), or adiposetissue development, and may result in leaky or non-functional vessels.Thus, one of the problems with administration of present chemotherapy orother tumor-destroying pharmaceutical drugs is that the pharmaceuticalsare not able to fully reach the entire tumor, due to this inadequateblood supply. However, the blood supply is typically sufficient enoughto allow the established tumor mass to continue to grow.

Further, the binding of HGF to the c-Met receptor is believed to be byway of a functional domain located in the N-terminal portion of themolecule. (Chirgadze et al., Nature Struct. Biol. 6:72 (1999); Schieringet al., PNAS 100(22):12654 (2003); Gherardi et al., 2003 Proc. Nat.Acad. Sci. USA 100:12039 Gherardi et al., 2006 Proc. Nat. Acad. Sci. USA103:4046.) HGF and HGF variants are described, for example, in U.S. Pat.Nos. 5,227,158; 5,316,921; and 5,328,837.

The practice of certain embodiments of the present invention willemploy, unless indicated specifically to the contrary, conventionalmethods of virology, immunology, microbiology, molecular biology andrecombinant DNA techniques within the skill of the art, many of whichare described below for the purpose of illustration. Such techniques areexplained fully in the literature. See, e.g., Sambrook, et al. MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al.Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A PracticalApproach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins,eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds.,1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A PracticalGuide to Molecular Cloning (1984).

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

Certain embodiments relate to methods, compositions and kits or alteringhepatocyte growth factor activity in a cell or plurality of cells. Acell generally indicates a single cell, whereas a plurality of cellsindicates more than one cell. The cells may comprise a tissue, organ orentire organism. Furthermore, the cell or cells may be located in vivo,in vitro, or ex vivo. Maintaining cell, tissue and organ cultures areroutine procedures for one of skill in the art, the conditions and mediafor which can be easily ascertained. (See, for example, Freshney,Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss 5^(th)Ed. (2005); Davis, Basic Cell Culture, Oxford University Press 2^(nd)Ed. (2002)).

In certain embodiments of the present invention, one or moreangiotensin-like factors may be used to identify agents that alterhepatocyte growth factor activity or c-Met receptor activity. Suchagents may inhibit or enhance signal transduction via an intracellularsignaling cascade, leading to cell proliferation. An agent that altershepatocyte growth factor activity or c-Met receptor activity may alterexpression and/or stability of HGF or c-Met, HGF or c-Met proteinactivity and/or the ability of HGF or c-Met to change phosphorylationstates of a substrate. Agents that may be screened within such assaysinclude, but are not limited to, antibodies and antigen-bindingfragments thereof, competing substrates or peptides that represent, forexample, a catalytic site or a dual phosphorylation motif, antisensepolynucleotides and ribozymes that interfere with transcription and/ortranslation of HGF or c-Met and other natural and synthetic molecules,for example small molecule inhibitors, that bind to and inactivate HGFor c-Met.

Candidate agents for use in a method of screening for an agent thatalters HGF activity or c-Met receptor activity according to certain ofthe present invention embodiments may be provided as a library orcollection of compounds, compositions or molecules. Molecules of such alibrary may typically include compounds known in the art as “smallmolecules” and having molecular weights less than 1,000 Daltons, and maybe less than 900, 800, 700, 600, 500, 400, or 300 Daltons, or any weighttherebetween. For example, members of a library of test compounds can beadministered to a plurality of samples, each containing at least one HGFor c-Met polypeptide as provided herein, and then assayed for theirability to enhance or inhibit HGF-mediated or c-Met-mediated binding toa substrate, or alteration in the phosphorylation state of a substrate.Compounds so identified as capable of influencing HGF or c-Met functionare valuable for therapeutic and/or diagnostic purposes, since theypermit treatment and/or detection of diseases associated with HGFactivity or c-Met activity. Such compounds are also valuable in researchdirected to molecular signaling mechanisms that involve HGF or c-Met,and to refinements in the discovery and development of future HGF orc-Met compounds exhibiting greater specificity.

Candidate agents further may be provided as members of a combinatoriallibrary, which preferably includes synthetic agents prepared accordingto a plurality of predetermined chemical reactions performed in aplurality of reaction vessels. For example, various starting compoundsmay be prepared employing one or more of solid-phase synthesis, recordedrandom mix methodologies and recorded reaction split techniques thatpermit a given constituent to traceably undergo a plurality ofpermutations and/or combinations of reaction conditions. Furthermore, anumber of computer algorithms available in the art allow for generationof members with desired characteristics relating to a particularmolecular structure, including a receptor. (See, for example, Pabo etal., Biochem., 25: 5987-5991 (1986); Lazar et al., Prot. Sci., 6:1167-1178 (1997); Lee, et al. Nature, 352: 448-451 (1991); Colombo etal., J. Am. Chem. Soc., 121: 6895-6903 (1999); Weiner, et al., J. Am.Chem. Soc., 106: 765-784 (1984)).

The resulting products comprise a library that can be screened followedby iterative selection and synthesis procedures, such as a syntheticcombinatorial library of peptides (see e.g., PCT/US91/08694,PCT/US91/04666) or other compositions that may include small moleculesas provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. Pat. No.5,798,035, U.S. Pat. No. 5,789,172, U.S. Pat. No. 5,751,629). Such alibrary is included in certain embodiments of the disclosed inventionand comprises multiple c-Met ligands, including agonists, partialagonists, and antagonists. Some of the ligands from the disclosedlibrary comprise peptides, as well as peptidomimetics and have exhibitedreceptor affinities in the sub-picomolar to nanomolar range.

Agents which alter HGF activity or c-Met activity may be identified bycombining a candidate agent with a HGF or c-Met receptor polypeptide ora polynucleotide encoding such a polypeptide, in vitro or in vivo, andevaluating the effect of the candidate agent on the HGF activity orc-Met activity using, for example, a representative assay describedherein.

For example, HGF activity or c-Met activity may be measured in wholecells transfected with a reporter gene whose expression is dependentupon the activation of an appropriate substrate. For example,appropriate cells (i.e., cells that express HGF or c-Met) may betransfected with a substrate-dependent promoter linked to a reportergene. In such a system, expression of the reporter gene (which may bereadily detected using methods well known to those of ordinary skill inthe art) depends upon activation of a substrate. Changes inphosphorylation states of a substrate may be detected based on a changein reporter activity. Candidate agents may be added to such a system, asdescribed above, to evaluate their effect on HGF activity or c-Metactivity.

Within other aspects, animal models are provided in which an animaleither does not express a functional HGF ligand or c-Met receptor, orexpresses an altered HGF ligand or c-Met receptor. Such animals may begenerated using standard homologous recombination strategies. Animalmodels generated in this manner may be used to study activities of HGFligand or c-Met receptor polypeptides and agents capable of altering HGFactivity or c-Met activity in vivo.

Certain embodiments as herein disclosed relate to an angiotensin-likefactor that is capable of specifically binding to a c-Met receptor in acell membrane. A cell membrane may include any cellular membrane, andtypically refers to membranes that are in contact with cytosoliccomponents, including intracellular membrane bounded compartments suchas mitochondrial inner and outer membranes, intracellular vesicles,vacuoles, ER-Golgi constituents, chloroplasts, other organelles and thelike, as well as the plasma membrane.

Angiotensin-like factors that are capable of specifically binding to ac-Met receptor include the angiotensin-like factors that react at adetectable level with the c-Met receptor, and may also react with anangiotensin receptor, but do not react detectably with receptorpolypeptides having unrelated sequences. In certain embodiments, theangiotensin-like factor reacts with an angiotensin II receptor. In otherspecific embodiments, the angiotensin-like factor does not react with anangiotensin II receptor. In some specific embodiments, theangiotensin-like factor reacts with an angiotensin IV receptor. In otherspecific embodiments, the angiotensin-like factor does not react with anangiotensin IV receptor.

The present disclosure further provides methods for identifying amolecule that interacts with, or specifically binds to, c-Met (or HGF).Such a molecule generally associates with c-Met (e.g. binds specificallyto the c-Met receptor) with an affinity constant (K_(a)) of at least10⁴, preferably at least 10⁵, more preferably at least 10⁶, still morepreferably at least 10⁷ and most preferably at least 10⁸. Affinityconstants may be determined using well known techniques. Methods foridentifying interacting molecules may be used, for example, as initialscreens for agents that are capable of altering HGF activity or c-Metreceptor activity, or to identify factors that are involved in the invivo HGF activity or c-Met activity. In addition to standard bindingassays, there are many other techniques that are well known foridentifying interacting molecules, including yeast two-hybrid screens,phage display and affinity techniques. Such techniques may be performedusing routine protocols, which are well known to those having ordinaryskill in the art (see, e.g., Bartel et al., In Cellular Interactions inDevelopment: A Practical Approach, D. A. Harley, ed., Oxford UniversityPress (Oxford, UK), pp. 153-179, 1993). Within these and othertechniques, candidate interacting proteins (e.g., putative HGFsubstrates or c-Met substrates) may be phosphorylated prior to assayingfor interacting proteins.

Certain embodiments of the disclosed invention comprise anangiotensin-like factor which may include a native angiotensinogenprecursor, a native angiotensin amino acid sequence or a fragment or afunctional subunit thereof. A fragment of any particular amino acidsequence may include at least one component of a larger amino acidsequence whether or not such fragment is functional. A functionalsubunit includes at least one component of a larger amino acid sequencethat retains a function compared to the larger amino acid sequence fromwhich it was derived. The function of the functional subunit may be thesame as the larger sequence, or it may be different in magnitude, inoutcome, etc. Thus, functional subunits may include molecules thatfunction as wild-type molecules, dominant negative molecules, orconstitutively active molecules.

In addition, encoding polynucleotides or polypeptide variants of anangiotensin-like factor may contain, respectively, one or morenucleotide or amino acid substitutions, additions, deletions, and/orinsertions relative to a native (e.g. wildtype, or a predominant ornaturally occurring allelic form). In some embodiments, a variantcomprises a molecule in which the N-terminal L-amino acid is replacedwith a D-amino acid. In certain embodiments, a variant comprises amolecule in which the N-terminal alpha amino acid is replaced with abeta or gamma amino acid. Variants preferably exhibit at least about75%, 78%, 80%, 85%, 87%, 88% or 89% identity and more preferably atleast about 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to a portionof a native polypeptide sequence or of a polynucleotide sequence thatencodes such a native molecule (for example, native HGF, c-Met receptor,or angiotensin-like factor). The percent identity may be readilydetermined by comparing sequences of the polypeptide or polynucleotidevariants with the corresponding portion of a full-length polynucleotideor polypeptide. Some techniques for sequence comparison include usingcomputer algorithms well known to those having ordinary skill in theart, such as Align or the BLAST algorithm (Altschul, J. Mol. Biol.219:555-565, 1991; Henikoff and Henikoff, PNAS USA 89:10915-10919,1992), which is available at the NCBI website (see [online]Internet:<URL: http://www/ncbi.nlm.nih.gov/cgi-bin/BLAST). Defaultparameters may be used.

Additionally, it has been discovered that rotatability of the chemicalbond, for instance, between the hydrophobic and basic amino acids in thegeneral formula described above, may determine whether a particularangiotensin-like factor acts as an agonist or as an antagonist for thec-Met receptor. In particular, as provided herein a number ofangiotensin-like factors comprise peptides or polypeptides having one ormore reduced peptide bonds, which are known to confer greater freedom ofrotation to the atoms on either side of the bond than is permitted by aconventional naturally occurring peptide bond. As such, and according tonon-limiting theory, the presence of one or more reduced peptide bondsin certain embodiments of the presently described angiotensin-likefactors provides flexibility at the molecular level that may enhance theability of these angiotensin-like factors to interact with c-Metreceptor binding sites for HGF and/or with HGF hinge regions. Furtheraccording to theory, the presence and location of the reduced peptidebond(s) in an angiotensin-like factor may favor its activity as a c-Metreceptor antagonist. Accordingly, in certain preferred embodiments theangiotensin-like factor may comprise one or more reduced peptide bonds.Typically when such a reduced peptide bond is present in a presentlydisclosed angiotensin-like factor, it is notated as Ψ and comprises orconsists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II]

Furthermore, computer algorithms are available in the art that enablethe skilled artisan to predict the three-dimensional structure of aprotein (up to approximately 80 amino acids in size) in order toascertain functional variants of a particular polypeptide. For instance,variants wherein all or a portion of the three-dimensional structure isnot substantially altered by one or more substitution, addition,deletion and/or insertion. (See, for example, Bradley et al., Science309: 1868-1871 (2005); Schueler-Furman et al., Science 310:638 (2005)).In this way, one of skill in the art could readily determine whether aparticular polypeptide variant, fragment or functional unit thereof iscapable of binding a c-Met receptor, and/or is capable of alteringhepatocyte growth factor activity or c-Met activity, including suchspecific activities as inducing cell proliferation, inducing cellscattering, inducing cell migration, inducing dysregulation of DNArepair, inducing chromosomal repair, inducing cell proliferation,inducing extracellular matrix disruption, inducing dysregulation ofapoptosis, inducing cellular extravasation, inducing expression of anadhesion molecule, inducing expression of an angiogenesis-relatedmolecule, inducing neurite growth or axon guidance, inducing celldifferentiation, inducing bone regeneration, inducing tissue repair, andinducing activation of intracellular signaling molecules, such as Gab1.

As used herein, inducing may refer to either initiating the response oractivity, or up-regulating (i.e. increasing in a statisticallysignificant way) the response or activity once such response or activityis already underway, or both.

Certain encoding polynucleotides, or polypeptide variants, aresubstantially homologous to a portion of a native (e.g., a naturallyoccurring, predominant form) gene or peptide, respectively (for example,HGF, c-Met, or angiotensin-like factor). Single-stranded nucleic acidsderived (e.g., by thermal denaturation) from such polynucleotide orpolypeptide variants are capable of hybridizing under moderatelystringent conditions to a naturally occurring DNA or RNA sequenceencoding a native polypeptide (or a complementary sequence). Apolynucleotide that detectably hybridizes under moderately stringentconditions may have a nucleotide sequence that includes at least 10consecutive nucleotides, more preferably 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotidescomplementary to a particular polynucleotide. In certain preferredembodiments such a sequence (or its complement) will be unique to apolypeptide for which alteration of expression is desired, and incertain other embodiments the sequence (or its complement) may be sharedby HGF and/or an angiotensin-like factor, and/or c-Met receptor.

Suitable moderately stringent conditions include, for example,pre-washing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);hybridizing at 50° C.-70° C., 5×SSC for 1-16 hours (e.g., overnight);followed by washing once or twice at 22-65° C. for 20-40 minutes withone or more each of 2×, 0.5× and 0.2×SSC containing 0.05-0.1% SDS. Foradditional stringency, conditions may include a wash in 0.1×SSC and 0.1%SDS at 50-60° C. for 15-40 minutes. As known to those having ordinaryskill in the art, variations in stringency of hybridization conditionsmay be achieved by altering the time, temperature, and/or concentrationof the solutions used for pre-hybridization, hybridization, and washsteps. Suitable conditions may also depend in part on the particularnucleotide sequences of the probe used, and of the blotted nucleic acidsample. Accordingly, it will be appreciated that suitably stringentconditions can be readily selected without undue experimentation when adesired selectivity of the probe is identified, based on its ability tohybridize to one or more certain sequences while not hybridizing tocertain other sequences.

Preferred embodiments of the disclosed invention includeangiotensin-like factors that are capable of binding specifically to acell surface c-Met receptor. Once bound, a given angiotensin-like factormay act as an agonist, a partial agonist, or an antagonist. Generally,it is understood in the art that a c-Met receptor agonist refers to anymolecule that mimics the effect of HGF to alter a biological activitymediated by the c-Met receptor, and specifically changes the function orexpression of the receptor, or alters the efficiency of signalingthrough the c-Met receptor, thereby altering (i.e. increasing ordecreasing in a statistically significant manner) an existing biologicalactivity or triggering a new biological activity, such as thosedescribed herein. For example, angiotensin-like factors capable ofacting as agonist ligands to the c-Met receptor may supportc-Met-mediated cell behavior such as cellular proliferation, tubularmorphogenesis, and/or cell migration. In vivo, c-Met agonists mayfacilitate wound healing, enhance angiogenesis, increase cognitivefunction or participate in mechanisms that mediate other physiologicalprocesses.

Alternatively, a c-Met receptor antagonist may generally refer to anymolecule that partially or fully blocks, inhibits or neutralizes abiological activity mediated by a c-Met receptor, by preventing thebinding of another ligand (such as HGF) to the c-Met receptor orotherwise interfering with the signaling of the receptor, therebysubstantially blocking, e.g., inhibiting at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more, of thebiological activity mediated by the receptor in the absence of theantagonist.

Certain embodiments relate to methods and compositions (and kits) fordiagnosing, preventing or treating a subject with a condition associatedwith c-Met receptor dysregulation. Certain embodiments contemplate sucha subject which may refer to any organism to be treated (eitherpreventatively or responsively), or diagnosed by the compositions and/ormethods disclosed herein. Such organism may include, but need not belimited to a vertebrate, including a mammal, including a human; a bird;a fish; an amphibian; and a reptile.

Certain embodiments relate to nucleic acids that encode the polypeptidescontemplated herein, for instance, angiotensin-like factor polypeptides.As one of skill in the art will recognize, a nucleic acid may refer to asingle and/or a double stranded DNA, cDNA or RNA in any form, and mayinclude a positive and a negative strand of the nucleic acid whichcomplement each other, including anti-sense DNA, cDNA and RNA. Alsoincluded are siRNA, microRNA, RNA-DNA hybrids, ribozymes, and othervarious naturally occurring or synthetic forms of DNA or RNA.

Certain embodiments include nucleic acids contained in a vector. One ofskill in the art can readily ascertain suitable vectors for use withcertain herein disclosed embodiments. A typical vector may comprise anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked, or which is capable of replication in a hostorganism. Some examples of vectors include plasmids, viral vectors,cosmids, and others. Some vectors may be capable of autonomousreplication in a host cell into which they are introduced (e.g.bacterial vectors having a bacterial origin of replication and episomalmammalian vectors), whereas other vectors may be integrated into thegenome of a host cell upon introduction into the host cell and therebyreplicate along with the host genome. Additionally, some vectors arecapable of directing the expression of genes to which they areoperatively linked (these vectors may be referred to as “expressionvectors”). According to related embodiments, it is further understoodthat, if one or more agents is co-administered to a subject, that eachagent may reside in separate or the same vectors, and multiple vectors(each containing a different agent the same agent) may be introduced toa cell or cell population or administered to a subject.

In certain embodiments, the nucleic acid of interest may be operativelylinked to certain elements of a vector. For example, polynucleotidesequences that are needed to effect the expression and processing ofcoding sequences to which they are ligated may be operatively linked.Expression control sequences may include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e. Kozak consensus sequences); sequences thatenhance protein stability; and possibly sequences that enhance proteinsecretion. Expression control sequences may be operatively linked ifthey are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest.

In addition to vectors, certain embodiments relate to host cells thatcomprise the vectors disclosed. One of skill in the art readilyunderstands that many suitable host cells are available in the art. Ahost cell may include any individual cell or cell culture which mayreceive a vector or the incorporation of nucleic acids and/or proteins,as well as any progeny cells. The term also encompasses progeny of thehost cell, whether genetically or phenotypically the same or different.Suitable host cells may depend on the vector and may include mammaliancells, animal cells, human cells, simian cells, insect cells, yeastcells, and bacterial cells. These cells may be induced to incorporatethe vector or other material by use of a viral vector, transformationvia calcium phosphate precipitation, DEAE-dextran, electroporation,microinjection, or other methods. For example, See Sambrook et al.Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring HarborLaboratory, 1989).

Certain embodiments also contemplate transgenic animals or transgenicplants comprising one or more nucleic acid molecule(s) such aspolynucleotides encoding one or more of an angiotensin-like factor, ac-Met receptor and HGF, that may be used to generate “knock-out” animalsor that may be used to produce angiotensin-like factors. Some examplesof transgenic animals that may be used include goats, cows, horses,pigs, rats, mice, rabbits, hamsters, or other animals. The transgenicanimals may be chimeric, non-chimeric heterozygotes, or non-chimerichomozygotes. (See, for example, Hogan et al., Manipulating the MouseEmbryo: A Laboratory Manual 2 ed., Cold Spring Harbor Press (1999);Jackson et al., Mouse Genetics and Transgenics: A Practical Approach,Oxford Univ. Press (2000); Pinkert, Transgenic Animal Technology: ALaboratory Handbook, Academic Press (1999).)

Other Therapeutic Agents

In certain embodiments, the present invention provides for administeringa second compound comprising at least one therapeutic agent inconjunction with a first compound comprising an angiotensin-like factor.The therapeutic agent may comprise a chemotherapeutic agent, a nucleicacid disrupting agent (for example, a DNA or RNA disrupting agent), aproliferative or anti-proliferative agent, radiation therapy includingone or more radiotherapy compounds, a cytotoxic agent, an adhesionmolecule (such as an integrin), an anti-inflammatory agent, a cytokine,an antibody, a polypeptide, a polynucleotide, a pro-apoptotic agent, ahormone, a statin, a growth factor, any combination of these or othertherapeutic agents.

In certain aspects, synergistic benefits may include the ability to usea lesser dosing or schedule frequency due to higher efficacy of the oneor more therapeutic agent; the ability to use the one or moretherapeutic agent for a longer duration in the regimen (over days,months or years) due to lower dose needed; the ability to use different,less toxic, therapeutic agents due to increased efficacy; the ability toincrease survival of a subject due to reduced toxicity; the ability todecrease non-target tissue or organ damage due to reduced toxicity(including, for example, reduced toxicity to liver, kidneys, immunesystem or nervous system); and other benefits.

Any number of chemotherapeutic agents may be used with the presentlydescribed embodiments in conjunction with administration of anangiotensin-like factor. For example, alkylating agents, such asmelphalan (C₁₃H₁₈C₁₂N₂O₂) and mitoxantrone; anthracyclines, such asdoxorubicin, idarubicin, epirubicin, and daunorubicin; cytokines, suchas Interferon-α, Interferon-β, TNF-α, IL-4; monoclonal antibodies(regardless of the source), such as Mylotarg (anti-CD33), anti-CD95,anti-CD20, anti-CD20/yttrium 90, anti-CEA, anti-HER-2, anti-CD20/iodine131, IgG2a, anti-CD52, anti-CD25, anti-CD30, anti-p185^(neu), anti-VEGF,anti-EGR, anti-HER/neu-anti-Fc gamma RI, anti-CTLA-4; statins; vitamins,such as vitamin C (ascorbic acid), super oxide dismutase (SOD-1) orother antioxidants; nucleoside analogs, such as cytosine arabinoside,cyclosporine; rapamycin; silominus; cytoxan; mitoxanthrone; steroids;gemcitabine; proteasome inhibitors, such as Bortezomib® (also calledVelcade®)[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronicacid; MG-132; radiation; taxanes, such as paclitaxel and docetaxel;vinca alkaloids, such as vincristine; Arsenic Trioxide;cis-diaminodichroplatanim (II); bleomycin (C₅₅H₈₄N₁₇O₂₁S₃); Bcl-2;Tamoxifen (C₂₆H₂₉NO); cisplatin; alitretinoin; anastrozole;azathioprine; bicalutamide; Trichostatin A, Trichostatin C, Oxamflati,Trapoxin A, FR901228, Apicidin, HC-Toxin, WF27082, Chlamydocin,Salicylihydroxamic acid, Suberoylanilide Hydroxamic Acid, AzelaicBishydroxamic acid, MW2796, MW2996, 6-(3-chlorophenylureido)carpoicHydroxamic acid, sodium butyrate, isovalerate, valerate,4-phenylbutyrate, phenylbutyrate, propionate, butrymide, isobutyramide,phenylacetate, 3-bromopropinate, tributyrin, busulfan; capecitabine;carboplatin; cyclophosphamide; cytarabine; etoposide; exemestane;finasteride; fluorouracil; fulvestrant; gemtuzumab ozogamicin;hydroxyurea; ibritumomab; ifosfamide; imatinib; letrozole; megestrolacetate; methotrexate; mifepristone; temozolomide; tretinoin;triptorelin; vinorelbine, histone deacetylase inhibitors (HDACs),including sodium valproate, and HDAC class I, class II, class III orothers; VEGF receptor ligands; PDGF receptor ligands; Kit inhibitors;Ret inhibitors; AMG706 (available from Amgen, Inc.); EGF receptorligands; ZD6474 (available from AstraZeneca); MP-412A (available fromAveo Pharmaceuticals, Inc.); serine/theronine inhibitors; RAFinhibitors; FLT-3 ligands; sorafenib (available from Bayer Corp.); BAY43-9006 (available from Onyx Pharmaceuticals, Inc.); SRC/ABL kinaseinhibitors (including dasatinib, available from Bristol-Myers Squibb);trk inhibitors (including lestaurtinib, available from Cephalon, Inc.);HER2 inhibitors, EphB4 receptor tyrosine kinase inhibitors (includingXL647, available from Exelixis, Inc.); FGF receptor ligands; XL999(available from Exelixis, Inc.); ErbB-2 inhibitors (such as lapatinib,available from GlaxoSmithKline); c-Kit inhibitors (including MLH518available from Millennium Pharmaceuticals, Inc.); PKC inhibitors(including PKC412, available from Novartis); STI571 (available fromNovartis); AMN107 (available from Novartis); AEE788 (available fromNovartis); OSI-930 (available from OSI Pharmaceuticals); OSI-817(available from OSI Pharmaceuticals); CSF-1R inhibitors, such assunitinib maleate and SU11248, both available from Pfizer; VDGF-2inhibitors, including AG-013736, available from Pfizer; SIR2 familymembers, such as Hst1p and Sir2p, Sir3p, and Sir4p; MICA or MICBligands; statins; demethylating agents (including decitabine and5-deazacytidine); gemcitabine; adjuvants; anti-sense RNA or DNA; siRNA;ribozymes or other enzymes; oligonucleotides, including DNA, RNA or anycombination thereof; cancer vaccines; or others.

In certain embodiments of the invention, a polynucleotide, polypeptide,cytokine (or cytokine inhibitor), or hormone (or hormone blocker) may bea therapeutic agent. Some non-limiting examples include, COX-2,Epidermal Growth Factor (EGF), Erythropoietin (“EPO”), Factor IX, FactorVII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), G-CSF,GM-CSF, Insulin, Insulin-like Growth Factor (IGF), interferons (e.g.,interferon-α, interferon-β, interferon-γ, interferon-ε, interferon-ζ,interferon-η, interferon-κ, interferon-λ, interferon-τ, interferon-ζ,interferon-ω), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), leukemia inhibitoryfactor, Neutrophil inhibitory factor (NIF), oncostatin M, Matrixmetalloproteinases (e.g. MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6,MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, etc.), PDGF, SCF,Soluble complement receptor 1, Soluble I-CAM 1, Soluble interleukinreceptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), solubleadhesion molecules, Soluble TNF receptor, Tissue plasminogen activator,Tumor necrosis factor β (TNF β), Tumor necrosis factor receptor (TNFR),Tumor necrosis factor-α (TNF α), VEGF, Erb, ERbB2, farnesyl proteintransferase, or others or inhibitors of any of these. As is evident fromthe foregoing, any anti-proliferating, pro-apoptotic, cytotoxic,anti-angiogenic or anti-neoplastic agent may be used according tocertain embodiments of the present invention. Alternatively, inhibitorsof such agents, or agents that promote or favor angiogenesis, cellproliferation, cell motility, etc. may be used in other embodiments ofthe invention, depending on the desired outcome.

Methods for Diagnosis, Prevention and Treatment

Certain embodiments provided herein are directed to assay and diagnosticmethods to identify agents useful to detect, prevent or treat acondition associated with c-Met dysregulation. Potential candidateagents may be screened according to assays described herein and may beperformed on cell samples that are in vivo, in vitro, or ex vivo.Included in these particular embodiments are cell samples, ranging froma single cell or cell contents, to multiple cells, tissues or organs.Any assay that measures the ability of an angiotensin-like factor tointeract with a cell or cell contents may be used for a diagnostic assayas described herein. For example, Western blot, ELISA, immunoassay,immunoprecipitation, cross-linking, chromatography, two-hybrid system,RIA, tissue immunohistochemistry, Northern blot, mass spectrometry(including MS-MALDI), IR, x-ray crystallography, immunoblot, FACS, laserscanning imaging, binding to a solid support (such as beads, arrays,columns, etc.) PCR, RT-PCR, or other methods are contemplated.

These diagnostic methods may also be used to determine if a particularcell, tissue, organ, or bodily fluid contains high (e.g. relative in astatistically significant manner, to the level in a control sample froma subject known to be free of c-Met dysregulation) levels of c-Met,which may be indicative of outcome for a subject.

Certain of the presently disclosed invention embodiments includepreventative treatment of a subject or cells, tissues or organs of asubject, that is suspected of having or susceptible to a conditionassociated with c-Met dysregulation. The preventative treatment may bethe same as or different from the regimen (dosing and schedule, as wellas choice of angiotensin-like factor and/or other therapeutic agents)employed to treat a subject or cells, tissues or organs of a subjectthat has been confirmed to have a condition associated with c-Metdysregulation. Prevention and/or treatment may also include the use ofvaccines comprising compositions disclosed herein, for example by way ofillustration and not limitation, one or more angiotensin-like factorsand/or c-Met-specific antibodies.

A condition associated with c-Met dysregulation includes any disorder orcondition in which underactivity, overactivity or improper activity of ac-Met cellular or molecular event is present. A subject having such adisorder or conditions would benefit from treatment with a compositionor method of the presently described embodiments. Some conditionsassociated with c-Met dysregulation include chronic and acute disordersand diseases, such as those pathological conditions that predispose thesubject to a particular disorder. Alternatively, a condition associatedwith c-Met dysregulation may also be non-pathological (such as, forexample, benign skin outgrowths, pregnancy, repair of broken bones orteeth, hair growth or re-growth).

Some non-limiting examples of conditions associated with c-Met receptordysregulation include hyperproliferative disorders, which refer tostates of activated and/or proliferating cells (which may also betranscriptionally overactive) in a subject including tumors, neoplasms,cancer, inflammatory conditions or disease, deposition of adiposetissue, etc. In addition to activated and/or proliferating cells, thehyperproliferative disorder may also include an aberration ordysregulation of cell death processes, whether by necrosis or apoptosis.Such aberration of cell death processes may be associated with a varietyof conditions, including inflammatory diseases, viral infections(whether the subject shows symptoms of a viral infection or not), cancer(including primary, secondary malignancies as well as metastasis) andother conditions (such as osteoarthritis and atherosclerosis). Otherconditions associated with c-Met receptor dysregulation includearthritis, diabetes, diabetic retinopathy, macular degeneration andobesity.

According to certain embodiments, virtually any type of cancer may betreated through the use of compositions and methods (and kits) disclosedherein, including but not limited to, organ cancers (e.g. cancer of theliver, kidney, or brain, glioblastoma or other cancer of the nervoussystem, stomach, ovarian, breast, prostate, other uro-genital, colon orother gastrointestinal, heart, lung, nasopharyngeal, skin, eye, oral,bone, or connective tissue cancer, etc.), as well as hematologicalcancers (T or B cell lymphomas, myeloma, leukemia, multiple myeloma,acute myeloid leukemia) are considered. Furthermore, “cancer” may referto any accelerated proliferation of cells, including solid tumors,ascites tumors, blood or lymph or other malignancies; connective tissuemalignancies; metastatic disease; minimal residual disease followingtransplantation of organs or stem cells; multi-drug resistant cancers,primary or secondary malignancies, angiogenesis related to malignancy,or other forms of cancer. Also contemplated by the presently claimedinvention are specific embodiments wherein only one of the above typesof disease are included, or specific embodiments are not included.

Other conditions associated with c-Met dysregulation include any numberof inflammatory and other diseases, including cancer, cirrhosis, AIDS,bacterial or viral infections, endometriosis, inflammatory jointdisease, such as arthritis (including osteoarthritis and rheumatoidarthritis), autoimmune disease, allergies (including irritation of skin,mucous membranes, bronchial airways, gastrointestinal tract, or othertissues as a reaction to foods, airborne or water borne irritants,skin-contacted irritants or other agents), asthma, dermatitis,vasculitis, sequelae of stem cell transplantation, includinggraft-versus-host diseases, organ transplantation, ophthalmologicdiseases (such as glaucoma, retinitis, diabetic retinopathy, uveitis,ocular photophobia, macular degeneration), pain associated with any ofthese, and any combination of these conditions.

Conditions associated with c-Met receptor dysregulation may also includeautoimmune diseases, which generally refer to any disease state governedby an abnormal immune response or a self-destructive immune response.Autoimmune disease is associated with a chronic inflammatory condition,with activated immune cells and a persistent inflammatory response thatmay include infiltration of mononuclear cells, proliferation offibroblasts, increased connective tissue and blood vessels, pain andtissue destruction. Various autoimmune diseases are well known andwell-characterized, for example, rheumatoid arthritis, systemic lupuserythematosus (SLE), Hashimoto's thyroiditis, autoimmune hemolyticanemia, Celiac Sprue, kidney disease (including acute renal failure);Alzheimer's Disease; Huntington's Disease; other neuroinflammation;peripheral neuropathy; inflammatory lung disease; chronic fatiguesyndrome, fibromyalgia, scleroderma, type I diabetes, ulcerativecolitis, lichen planus, autoimmune hepatitis, Berger's disease,idiopathic thrombocytopenia purpura, rheumatic fever, Graves' disease,chronic fatigue syndrome, multiple sclerosis, vitiligo, perniciousanemia, type I diabetes, pemphigus, polymyositis, dermatomyositis,myasthenia gravis, Crohn's disease, ulcerative colitis, inflammatorybowel disease, psoriasis, dermatitis, scleroderma, Sjogren syndrome,meningitis, encephalitis, uveitis, eczema, respiratory distresssyndrome, Reynaud's syndrome, glomerulonephritis, microglialmalfunction, chronic infiltrating lung diseases, as well as immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousanemia (Addison's disease), multiple organ injury syndrome, hemolyticanemia, and others. Also contemplated by the presently claimed inventionare specific embodiments wherein only one of the above conditions ordiseases are included, or any one or more of the specific conditions ordiseases are not included.

Another method of treatment or prevention contemplated by certainembodiments of the present invention includes administering acomposition that comprises a desired nucleic acid molecule such that itstably integrates into the chromosome of certain desired cells. Forexample, such compositions may be integrated into tumor cells or immunesystem cells in order to regulate growth and/or antibody production.

In other embodiments, the condition associated with c-Met dysregulationincludes alteration of angiogenesis. Angiogenesis generally refers togrowth of blood vessels or lymphatic vessels. The presently disclosedcompositions and methods include those that may find use in diagnosis,prevention and/or treatment of conditions associated with the need forincreased angiogenesis, or decreased angiogenesis. In certain instances,increased angiogenesis is desired, such as diabetes, limb perfusion,hepatic disease, renal disease, neurodegeneration, preeclampsia,respiratory distress, organ transplant, bone or tooth regeneration, hairre-growth, wound or tissue healing, fetal or placental abnormalities,infertility or other reproductive dysfunction, central nervous systemimpairment or repair, and augmenting or restoring blood flow to ischemictissues, such as heart or brain following myocardial infarction orstroke. Alternatively, in other instances, decreased angiogenesis isdesired, such as for tumor growth or metastasis, diabetic retinopathy,reduction of adipose tissue or prevention of deposition of adiposetissue, or in the field of reproductive biology or animal husbandry, inorder to reduce the number of developing embryos in a pregnant subject,as an abortifacient, or as a means to achieve a biochemical infertilityor castration of a subject.

The compositions and methods of treatment described herein generallyrefers to both therapeutic treatment and prophylactic or preventivetreatment, wherein the object is to prevent or lessen an undesiredphysiological change or disorder, such as the development of a disease,including inflammatory disease or cancer, or reduction in anothercondition, such as loss of body fat or adipose tissue. Further,beneficial or desired clinical results may include, but not be limitedto, alleviation or amelioration of symptoms, diminishment of extent ofdisease, stabilized (not worsening) state of disease, delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether measurable orimmeasurable, whether detectable or undetectable.

In some embodiments of the presently disclosed invention, treatment mayrefer to prolonging survival of a subject compared to the expectedsurvival if the subject does not receive treatment. Some subjects thatmay be treated would have already been identified as having a conditionassociated with c-Met dysregulation. Other subjects that may be treatedmay be prone to or suspected of having a condition associated with c-Metdysregulation. Typically treatment may include, for example, a regimenthat involves administration of an angiotensin-like factor such as ac-Met receptor agonist, or antagonist, or both, at various stages of thedisease or conditions to be treated, as well as combination treatmentemploying such agonists or antagonists, along with other therapeuticagents. Preventative treatment may further involve measuring orquantitatively or qualitatively detecting levels of c-Met or HGF, and/ordetermining that the level of c-Met or HGF in a subject is abnormallyhigh, potentially diagnosing the subject as being at risk for having orsusceptible to or otherwise predisposed to a disease associated withhigh levels of c-Met or HGF, and implementing a corresponding treatmentregimen involving administration of one or more c-Met ligand(s) alone orin conjunction with administration of one or more other therapeuticagent(s). (For example, the angiotensin-like factor compositions may beboth neuroprotective as well as neuroregenerative).

In certain instances, the level of c-Met or HGF in a specific tissue,organ, tumor, or biological sample (including a bodily fluid, such asblood, lymph, ascites, urine, saliva, serum, plasma; a cell or pluralityof cells, a tissue or plurality of tissues; or an organ, or a pluralityof organs, or a biopsy or component of any of these) is about 2-3 timeshigher than the level of c-Met or HGF in a normal sample. In otherinstances, the level of c-Met or HGF may be about 3-5, 5-10, 10-100times or more the amount of c-Met or HGF in a normal sample.

As used herein, administration of a composition or therapy such as anangiotensin-like factor as herein disclosed, or a pharmaceuticalcomposition comprising an angiotensin-like factor, refers to deliveringthe same to a subject, regardless of the route or mode of delivery.Administration may be effected continuously or intermittently,systemically or locally. Administration may be for treating a subjectalready confirmed of having a disease or disease-state, or for subjectssusceptible to or at risk of developing a disease or disease-state.Co-administration, includes both simultaneous and/or sequential deliveryof multiple agents in any order and on any dosing schedule.

An effective amount of a therapeutic or pharmaceutical compositionrefers to an amount sufficient, at dosages and for periods of timeneeded, to achieve the desired clinical results or beneficial treatment,as described herein. An effective amount may be delivered in one or moreadministrations. If the administration is to a subject already known orconfirmed to have a disease or disease-state, the term “therapeuticamount” may be used in reference to treatment, whereas “prophylacticallyeffective amount” may be used to describe administrating an effectiveamount to a subject that is susceptible or at risk of developing adisease or disease-state as a preventative course.

Kits and Articles of Manufacture

In certain embodiments, the present invention relates to kits andarticles of manufacture relating to one or more compositions and/ormethods of the presently disclosure. Kits and/or articles of manufacturemay comprise components and/or compositions that are features of theherein disclosed embodiments, as well as packaging materials,instructions for using the components and/or methods, containers,buffers, additional detection reagents, as well as any apparatusnecessary for practicing the methods relating to such kits or articlesof manufacture.

In certain embodiments, a kit may be used to detect hepatocyte growthfactor activity and/or c-Met activity. In certain embodiments, the kitmay be used to diagnose a condition associated with c-Met dysregulation.The kit may comprise an indicator, which may be a calorimetric marker, anumber, or a computerized sound for indicating the presence oralteration (increase or decrease) of hepatocyte growth factor activityor c-Met receptor activity in a biological sample. The kit may furthercomprise an apparatus or device for administering the angiotensin-likefactor to the biological sample.

In other embodiments, a kit may be used to treat a condition associatedwith c-Met dysregulation in a subject or biological sample, wherein thekit comprises a first compound comprising an angiotensin-like factorthat alters activity of hepatocyte growth factor, and wherein theangiotensin-like factor is capable of specifically binding to a c-Metreceptor in a cell membrane. The kit may also contain an apparatus ordevice for administering the angiotensin-like factor to the subject orbiological sample.

Pharmaceutical Compositions

In certain embodiments of the disclosed invention, a pharmaceuticalcomposition comprising at least one herein disclosed composition, suchas an angiotensin-like factor as herein disclosed, or a pharmaceuticalcomposition comprising an angiotensin-like factor is administered to asubject. As used herein, a pharmaceutical composition generally refersto the combination of an active pharmaceutical drug or other therapeuticagent and an excipient or carrier, whether inert or active, wherein thepharmaceutical composition comprises at least one angiotensin-likefactor that is suitable for therapeutic use, including prophylactic use,in vivo, in vitro, or ex vivo.

In certain embodiments, the present invention relates to formulations ofone or more compositions disclosed herein in pharmaceutically-acceptableexcipients or carriers for administration to a cell or a subject eitheralone, or in combination, with one or more other modalities of therapy.It is understood that, if desired, a composition as disclosed herein maybe administered in combination with other agents as well, includingtherapeutic agents. Such compositions may be synthesized de novo orpurified from host cells or other biological sources.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable excipients orother carriers, and may contain acceptable salts. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g. salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g. sodium,potassium, lithium, ammonium, calcium and magnesium salts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions as described herein(e.g., pharmaceutical compositions that comprise the presently disclosedangiotensin-like factor), the type of carrier will typically varydepending on the mode of administration. Compositions of the presentinvention may in certain embodiments be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,mucosal, intravenous, intratumor, rectal, parenteral, intraperitoneal,subcutaneous and intramuscular administration.

Carriers for use with such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the route of administration,the rate and expected duration of release and the nature of thecondition to be treated or prevented.

Certain embodiments of the invention may utilize an alkalinizing agent,which is typically soluble in aqueous phase under physiological pHconditions. Such alkalinizing agents are well known to those in the artand may include alkali or alkaline-earth metal hydroxides, carbonates,bicarbonates, phosphates, sodium borate, as well as basic salts (asdiscussed herein).

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsthat are herein disclosed. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and5,942,252. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte response in a host.

In another illustrative embodiment, calcium phosphate core particles areemployed as carriers, adjuvants, or as controlled release matrices forthe compositions of this invention. In certain embodiments, an adjuvantmay be necessary in order to increase the immune response of thesubject. Adjuvants are well known in the art and may include cytokines,dead viruses or bacteria or fragments thereof, antibodies, or any otheragent that heightens an immune response.

The pharmaceutical compositions as provided herein will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions described herein may beformulated as lyophilizates.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet. Thepharmaceutical composition may take the form of tablets, lozenges,pills, troches, capsules, elixirs, powders, granules, suspensions,emulsions, syrups, or tinctures. Slow-release or delayed-release formsmay also be prepared (for example, in the form of coated particles,multi-layer tablets or microgranules).

The compositions may also contain any of a variety of additionalcomponents, for example, pharmaceutically acceptable binders, such asgum tragacanth, gum acacia, sodium alginate, carboxymethylcellulose,polyethylene glycol, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite,agar, alginic acid and the like; a lubricant, such as magnesiumstearate; a sweetening agent, such as sucrose, lactose, glucose,aspartame or saccharin may be added; a diluent, such as lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate; a flavoring agent, such aspeppermint, oil of wintergreen, orange, raspberry, bubblegum, or cherryflavoring, coating agents, such as polymers or copolymers of acrylicacid and/or methacrylic acid and/or their esters, waxes, fatty alcohols,zein, shellac or gluten; preservatives, such as sodium benzoale, vitaminE, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben, orsodium bisulphate; lubricants, such as magnesium stearate, stearic acid,sodium oleate, sodium chloride or talc; and/or time delay agents, suchas glyceryl monostearate or glyceryl distearate.

In certain embodiments, a tablet or pill may be in the form of acompression coating or alternatively in the form of a spray coating. Acompression coating may include a small tablet or pill utilized as partof the compression of a second tablet and wherein the small tablet islocated nearly in the center of the rest of the powder compressedoutside. A spray coating may include an overcoating of a tablet with thecoating preparation containing an active substance.

In certain embodiments, the pharmaceutical compositions of the presentinvention include “slow-release” or “immediate release” forms. As usedherein, “slow-release” generally refers to a release of 20% to 60% in 1hour and greater than 70% in 6 hours or 40% to 80% in 2 hours, andgreater than 70% in 6 hours in 500 ml of water (HCl 0.1N) in USPapparatus 1 (37° C., 100 RPM). Whereas, “immediate release” generallyrefers to a release of more than 70% in 30 minutes, in 500 ml of water(HCl 0.1N) in USP apparatus 1 (37° C., 100 RPM).

In addition, certain embodiments of the disclosed invention includepharmaceutical compositions in the form of an oral tablet or pill. Suchoral formulations may include sections or discrete volumes that containan active drug. A section of a tablet may form, for example, a layer ofa multilayer tablet (i.e. a layer of a bilayer tablet) or a core of atablet or a coating fully or partially covering a core of a tablet. Asection may also be a particle fully or partially covered by a coatingor a coating fully or partially covering a particle. The oralformulations described may contain a “fast release” or “slow release”component, or one or more of each. In some embodiments, thepharmaceutical composition comprises multiple therapeutic agents, whichmay be separated into different sections. In certain embodiments, eachsection is substantially free of another agent. For example, one sectionof a tablet comprising an active agent (such as a c-Met receptor ligand)is substantially free of a second compound comprising anothertherapeutic agent. Substantially free generally refers to less than 10%,less than 5%, less than 3%, less than 1%, less than 0.5%, or less than0.2% by weight. Furthermore, in certain embodiments, a barrier sectionmay separate components or agents in the pharmaceutical drugformulation.

In certain embodiments, the tablet or pill weighs in the range of 100mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, and anyvalue therebetween or greater. The oral dosage formulations of certainembodiments of the present invention may be manufactured according toknown methods in the art, and may be packaged as known, including in amoisture and/or oxygen and/or light protective packaging material.

In addition, liquid forms of the pharmaceutical compositions may includea liquid carrier, such as water, oils (olive oil, peanut oil, sesameoil, sunflower oil, safflower oil, arachis oil, coconut oil), liquidparaffin, ethylene glycol, propylene glycol, polyethylene glycol,ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides,or mixtures thereof.

If the subject composition is administered parenterally, the compositionmay also include sterile aqueous or oleaginous solution or suspension.Suitable non-toxic parenterally acceptable diluents or solvents includewater, Ringer's solution, isotonic salt solution, 1,3-butanediol,ethanol, propylene glycol or polythethylene glycols in mixtures withwater. Aqueous solutions or suspensions may further comprise one or morebuffering agents, such as sodium acetate, sodium citrate, sodium borateor sodium tartrate. Of course, any material used in preparing any dosageunit formulation should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compounds maybe incorporated into sustained-release preparation and formulations.Dosage unit form, as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unit maycontain a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms arelargely dictated by and directly dependent on the unique characteristicsof the active compound and the particular therapeutic or prophylacticeffect to be achieved, as well as the limitations inherent in the art ofcompounding such an active compound for treatment in subjects. Exemplaryand non-limiting dosage ranges may be from 0.1-10 mg/kg, 1.0-20 mg/kg,5.0-50 mg/kg, 10-100 mg/kg, or any values therebetween.

Typically, these formulations will contain at least about 0.01% of theactive compound or more by weight of the active substance. However, thepercentage of the active ingredient(s) may be varied and mayconveniently be between about 1-99%, including about 60% or 70% or moreof the weight or volume of the total formulation. Naturally, the amountof active compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

For oral administration the compositions presently disclosed mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein topically, orally,subcutaneously, parenterally, intravenously, intramuscularly, or evenintraperitoneally. Such approaches are well known to the skilledartisan, some of which are further described, for example, in U.S. Pat.No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. Incertain embodiments, solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, and/orvegetable oils. Proper fluidity may be maintained, for example, by theuse of a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. The prevention of the action of microorganisms can befacilitated by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationspreferably meet sterility, pyrogenicity, and the general safety andpurity standards as required by FDA Office of Biologics standards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles,microencapsulation, lipid particles, vesicles, and the like, are usedfor the introduction of the presently disclosed compositions intosuitable host cells/organisms. In particular, such compositions may beformulated for delivery either encapsulated in a lipid particle, aliposome, a vesicle, a nanosphere, microsphere or microparticle, ananoparticle or the like. Alternatively, compositions disclosed hereincan be bound, either covalently or non-covalently, to the surface ofsuch carrier vehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art.Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol. Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, the use of liposomes does notappear to be associated with autoimmune responses or unacceptabletoxicity after systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way. To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 μm) may be designed using polymers able to be degraded invivo. Such particles can be made as described, for example, by Couvreuret al., Crit. Rev Ther Drug Carrier Syst. 1988; 5(1):1-20; zur Muhlen etal., Eur J Pharm Biopharm. 1998 March; 45(2):149-55; Zambaux et al. JControlled Release. 1998 Jan. 2; 50(1-3):31-40; and U.S. Pat. No.5,145,684.

Dosing Schedules

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions may be administered byinjection (e.g., intracutaneous, intramuscular, intravenous orsubcutaneous), intranasally (e.g., by aspiration) or orally. Suitabledosage formulations and methods of administering the agents are readilydetermined by those of skill in the art. In certain instances, theangiotensin-like factor compounds may be administered at about 1 mg/kg,5 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg 40 mg/kg, 45mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg or any valuetherebetween or greater. When the angiotensin-like factor compoundsdescribed herein are co-administered with at least one other therapeuticagent (such as a chemotherapeutic agent), a synergistic drug interactionmay occur that allows for an effective amount dosage to be less thanwhen the particular agent is administered alone.

If more than one angiotensin-like factor compound or more than onetherapeutic agent are co-administered, the agents may be administeredusing different modes, routes or different formulations, as well asdifferent dosing schedules. In addition, multiple same or differentangiotensin-like factor compounds and/or multiple same or differentother therapeutic agents may be combined in a particular dosingschedule.

In certain instances, angiotensin-like factor doses (and optionally, atleast one other therapeutic agent dose) may be provided between 1 dayand 14 days over a 30 day period. In certain instances, angiotensin-likefactor doses (and optionally, at least one other therapeutic agent dose)may be provided 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 daysover a 60 day period. Alternatively, dosing may be for about 2 weeks, 4weeks, 6 weeks or 8 weeks. Administering may comprise 1, 2, 3, 4, 5, 6,7, 8 or more cycles of treatment. Alternate protocols may be appropriatefor individual subjects. A suitable dose is an amount of a compoundthat, when administered as described above, is capable of altering orameliorating symptoms of a condition associated with c-Metdysregulation, or is at least 10-50% above the basal (i.e., untreated)level, which can be monitored by measuring specific levels of bloodcomponents, for example, cytokines, or pharmaceutical drug metabolites.

In general, dosage of any pharmaceutical drug depends on a variety offactors, including age, weight, gender, genetic background, route ofadministration, disease state, physical condition and other factors.Thus, suitable dosage sizes for the first compound comprising anangiotensin-like factor will vary with the subject, and can becalculated according to various formulas known in the art. In someregimens, only one dose will be administered to a subject per day, andin other regimens multiple doses will be administered in a single day.In addition, the dosing schedule may vary over a period of hours, days,weeks, months or years depending on the goal of the therapy and thedisease state of the subject. In certain embodiments, theangiotensin-like factor can be delivered prior to, concurrently with orsubsequent to the dosing of two or more therapeutic agents.

In addition, the dosage range of the first compound comprising anangiotensin-like factor may be co-administered with another therapeuticagent in accordance with certain embodiments of the invention. As usedherein, “co-administration” includes administration of the firstcompound substantially simultaneously with at least one othertherapeutic agent, or substantially sequentially (either prior to orsubsequent to) at least one second compound comprising a therapeuticagent. Thus, the dosage range for co-administration of the firstcompound with the second compound may be more than or less than thedosage for either compound alone. In certain embodiments of theinvention, a synergy results in co-administration, and a lower dosagerange for the first compound and/or the second compound would beemployed

In certain aspects, “co-administration” refers to administration ofeither the first compound or the second compound within about 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 120 minutes or less from the time ofadministering a dose of the other compound (either second compound orfirst compound, respectively). In other aspects, “co-administration”refers to administration of at least the first compound or the secondcompound within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 24, 48, 72hours or less from administering a dose of the other compound (eithersecond or first compound, respectively). In still other embodiments ofthe present invention, “co-administration” refers to administration ofthe first compound or the second compound within about 1, 2, 3, 4, 5, 6,7 days or less from the time of administering a dose of the secondcompound or first compound, respectively. Thus, in certain instances thefirst compound comprising an angiotensin-like factor may be administeredprior to, concurrently with, or subsequent to at least one secondcompound comprising another therapeutic agent. In some instances whenmore than one other therapeutic agent is administered to a subject, theangiotensin-like factor may be administered prior to, concurrently with,or subsequent to one or more or even all of the other therapeuticagents. In other instances the angiotensin-like factor may beadministered multiple times while the one or more other therapeuticagents are administered once only, or multiple times. If both theangiotensin-like factor and at least one other therapeutic agent areadministered multiple times, then they may be interspaced, or all of onecompound may be delivered prior to, concurrently with, or subsequent toone or more other compounds. Thus, any combination of dosing schedulesand regimen may be employed within embodiments contemplated according tothe present invention.

Also contemplated are multiple doses or dosing schedules, includingalternating administering the first compound comprising anangiotensin-like factor and a second compound comprising at least onetherapeutic agent, or alternating the first compound with multipletherapeutic agents. If such dosing schedule is employed, it isrecognized that “co-administration” of the first compound and the othertherapeutic agent(s) may include multiple rounds of administration, inany order of delivering the compounds or agents. The present inventionfurther contemplates that it may be necessary in certain embodiments toadminister to the subject a higher initial loading dose of the firstcompound to achieve peak blood levels, followed by lower maintenancedoses. Furthermore, one particular route of administration may becontinuous over the course of several minutes, hours or days, while theother therapeutic agent(s) or angiotensin-like factor(s) may beadministered by an alternate route or the same route, over the course ofseveral minutes, hours or days.

According to the present disclosure, the first compound comprising anangiotensin-like factor compound and at least a second compoundcomprising one other therapeutic agent may in some embodiments bedelivered to the subject by the same route of administration ordifferent routes of administration, regardless of whether or not theyare “co-administered” to the subject.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated subjects ascompared to non-treated subjects. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which areroutine in the art and may be performed using samples obtained from asubject before and after treatment.

The present disclosure also contemplates methods of modifying andderivatizing the herein disclosed angiotensin-like factor(s) to increasedesirable properties (for example, binding affinity, activity or thelike) or to minimize undesirable properties (such as nonspecificreactivity, toxicity and the like). The principles of chemicalderivatization are well understood. In some embodiments, iterativedesign and chemical synthesis approaches are used to produce a libraryof derivatized child components from a parent compound. In otherembodiments, rational design methods are used to predict and model insilico ligand-receptor interactions prior to confirming results byroutine experimentation.

EQUIVALENTS

While particular steps, elements, embodiments and applications of thepresent invention have been shown and described herein for purposes ofillustration, it will be understood, of course, that the invention isnot limited thereto since modifications may be made by persons skilledin the art, particularly in light of the foregoing teachings, withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

The following Examples are presented by way of illustration and notlimitation.

EXAMPLES Example 1 Materials and Methods

This Example describes materials and methods used in the Examples thatfollow and also in the generation of data for FIGS. 1-19; additionaldetail regarding experimental procedures and results can be found aboveunder “Brief Description of the Drawings”.

Compounds. Norleual (Compound 2, SEQ ID NO:43) was synthesized bySyngene (Bangalore, India) and purified by reverse phase HPLC. HGF (SEQID NO:83) was purchased from R&D systems (Minneapolis, Minn., USA).

Antibodies. Antibodies specific for c-Met (SEQ ID NO:84), Gab1, andphospho-tyrosine were purchased from Upstate Biotechnology Inc. (LakePlacid, N.Y., USA). Anti-phospho-tyrosine, HAM1676, was purchased fromR&D systems. Phospho-Akt- and Erk-specific antibodies were purchasedfrom Cell Signaling (Danvers, Mass., USA). GAPDH-specific antibody waspurchased from Biodesign (Saco, Me., USA).

Cell culture. Cell lines HEK293, MDCK, B16-F0 and B16-F10 (all availablefrom ATCC, Manassas, Va.) were grown in DMEM, 10% fetal bovine serum(FBS). Human Umbilical Vein Endothelial Cells (HUVEC) were grown inEGM-2 (Lonza, Basel Switzerland).

Iodination of ¹²⁵I-HGF and ¹²⁵I-Norleual. Carrier free HGF was iodinatedusing the chloramine T method described by Higuchi et al., 1991 Biochem.Biophys. Res. Commun. 176: 599-607. 10 μl of 1.5M NaPO₄ (pH 7.4), 4 μl˜1mCi Na¹²⁵I (Perkin Elmer, Waltham Mass., USA), 10 μl of 1 ng/ml HGF wereadded to 5 μl of 0.1 mg/ml chloramine-T which was added four times at 90second intervals and stopped by 50 μl of 50 mM N-acetyl-1-tyrosine, 200μl of 60 mM NaI, and 200 μl of 1.2 mg/ml urea. ¹²⁵I-HGF was separatedusing a G-25 Sephadex (1.5×20 cm) filtration column. Norleual wasiodinated using the chloramine-T method as described (Hanesworth et al.,1993 J. Pharmacol. Exp. Ther. 266: 1036-1042). The reaction wasperformed in 0.2 M sodium phosphate buffer (pH 7.2) containing a totalvolume of 118 μl (50 μl [1 ng/ml] Norleual, 50 μl additional buffer, 10μl chloramine T (4 mg/ml), and 8 μl consisting of 2 mCi Na ¹²⁵I (PerkinElmer)]. The reaction was incubated for 2 minutes at room temperature(RT) and stopped with 50 μl Na₂S₂O₅ (5 mg/ml). ¹²⁵I-Norleual waspurified by HPLC.

¹²⁵I-HGF binding assay. Mouse liver was homogenized in hypotonic bufferfortified with 0.1% bovine serum albumin as described by Hanesworth etal. (1993). 600 μg of membrane protein was incubated with 50 pM ¹²⁵I-HGFwith various concentrations of HGF or Norleual in a total volume of 100μl phosphate-buffered saline (PBS) fortified with 0.1% BSA. Bindingoccurred for one hour on ice. Membranes were pelleted by centrifugationand washed twice with incubation buffer. Radioactivity was counted witha gamma counter (Isomedic 10/880, ICN, Cost Mesa, Calif., USA). Specificbinding was calculated and normalized to control counts. Competitionfits by Prism software (GraphicPad, San Diego, Calif.) were performedaccording to the manufacturer's instructions.

¹²⁵I-Norleual binding assay. HEK293 cells were grown to confluency inDMEM fortified with 10% FBS. Media was aspirated and cells were washedtwice with ice-cold PBS. Cells were lysed with hypotonic buffer andhomogenized as described by Zhang et al. (1999, J. Pharmacol. Exp. Ther.289: 1075-1083; 2003 J. Cell Biochem. 88: 408-417). 450 μg of membraneprotein was incubated with 1.5 nM ¹²⁵I-Norleual with or without Norleualor HGF in a total volume of 250 μl PBS, 0.1% BSA. Tubes were spun andbinding was carried out for 60 minutes at RT. Bound and free ligandswere separated by vacuum filtration using (Brandel Cell Harvester,Schleicher & Schull, Keene, N.H., USA) 32 glass filters. Radioactivityretained by the filters was measured using a gamma counter. Total countswere normalized to the average of non-displaced ¹²⁵I-Norleual (control)and displayed as percent control. Data analysis was preformed usingPrism software (GraphicPad) per the supplier's recommendations.

Immunoprecipitation and Western blotting. Cells were serum starved fortwo hours in DMEM before treatment. Cells were harvested using RIPAlysis buffer (Upstate Biotechnol. Inc., Lake Placid, N.Y.) withphosphatase inhibitor cocktails 1 and 2 (Sigma, St Louis, Mo., USA).Protein concentration was determined using the BCA assay (Pierce,Rockford, Ill., USA). Lysates were incubated with antibody overnight at4° C. Immunoprecipitation controls received mouse non-specific Ig(Upstate). Proteins were captured with protein-A agarose (Upstate) andwere washed with PBS. Proteins were resolved using SDS-PAGEelectrophoresis (Criterion, BioRad, Hercules, Calif., USA), transferredto nitrocellulose, and incubated with appropriate antibodies. Proteinswere visualized using the Supersignal West Pico ChemiluminescentSubstrate system (Pierce). The film images were digitized and analyzedusing TotalLab® software (AmershamPharmacia/GE HealthCare, Piscataway,N.J.).

Aortic ring assay. 48-well plates were coated with Growth Factor ReducedMatrigel (BD Biosciences, San Jose, Calif., USA, thick gel method).Under microscopic dissection, the thoracic aorta of a 6 week-old C57mouse was cut into 0.5 mm sections, washed in PBS, and placed in theMatrigel-coated wells. The rings were incubated for 4 days ingrowth-factor-containing EGM-2 (Lonza Biosciences, Rockland, Me. andBasel, Switzerland) with or without Norleual at 37° C. in 5% CO₂/air.Photos were taken and angiogenic area was measured using Image J (ImageProcessing and Data Analysis in Java, available from NIH, Bethesda,Md.). Areas were normalized to average control area.

Scattering assay. MDCK cells were grown to confluency on coverslips. Thecoverslips were transferred to fresh plates and Norleual, HGF, and/orvehicle were added. Plates were incubated at 37° C. with 5% CO₂ for 48hours. Media was removed and cells were methanol fixed. Cells werestained with Diff-Quik Wright-Giemsa (Dade-Behring, Newark, Del., USA).Digital images of each coverslip sector (top, bottom, left, and right)were acquired using a Zeiss Axiovert 40 inverted microscope. Scatteringwas scored in blinded fashion by comparison to a set of calibrationscattering images by two investigators. The scores for each cover slipwere averaged and HGF and HGF+Norleual coverslip scattering wasnormalized to control scattering.

Scratch Assay. Confluent B16-F10 cells in 48 well plates were scratchedwith a 200 μl pipette tip. Wells were treated with vehicle, HGF, and/orNorleual. Photos were taken at various time points. Photos were gradedby two investigators.

Primary tumor melanoma cancer model. Six-month old male C57BL/6 micewere anesthetized and 500 000 B16-F0 cells were injected subcutaneously,and a slow release ethylene vinyl acetate, Elvax, drug pellets (DuPont,Wilmington, Del., USA) containing Norleual or BSA were surgicallyimplanted intramuscularly into the gluteus maximus at a dose of 21μg/kg/day. Tumors were measured with digital calipers every two to threedays and volumes were calculated using the equation: [V=(4/3)*pi*r³].Upon daily visual assessment, appropriate measures were taken ifwarranted (adhering to the guidelines of Part 3 of the Washington StateUniversity Guide for the Care and Use of Laboratory Animals).

In vivo metastasis. Six to eight month old C57BL/6 mice were injectedwith 400 000 B16-F10 cells in 200 μl PBS by tail vein injection. Two tothree weeks later, mice were anesthetized and lungs were perfused withPBS and removed. Photos were taken and lungs were solubilized in 1%Triton x-100, 20 mM Tris, 0.15 M NaCl, 2 mM EDTA, and 0.02% sodiumazide. Samples were disrupted by sonicaton (Mixonix, Farmingdale, N.Y.,USA) and spun. The supernate was transferred to a 96 well plate andmelanin absorbance at 410 nm was measured using a plate reader (BiotekSynergy2, Winooski, Vt., USA).

Statistics. Independent one-way analysis of variance (ANOVA)s (InStatv.3.05, GraphPad and Prism) were used to determine differences amonggroups. Tukey-Kramar or Bonferroni's multiple comparison post-hoc testswere performed where necessary. Statistical comparisons of two groupswere determined using the two-tailed Student's t-test (InStat and Prism)with a level of significance of 0.05.

Example 2 The Angiotensin-Like Factor Norleual Competes with HGF forBinding to the C-Met Receptor

This Example shows that the AT₄ receptor antagonist, Norleual (Compound2, SEQ ID NO:43, [Nle-Y-L-Ψ-(CH₂—NH₂)—H—P—F], Davis et al., 2006Neuroscience. 137: 1369) competed with HGF for binding to c-Met on cellsurfaces and in a cell membrane. Materials and methods were as describedin Example 1. In separate tests of unlabeled competitors, both Norleualand HGF competed with ¹²⁵I-HGF for high affinity binding to mouse liverplasma membranes (FIGS. 2A, 2B). The IC₅₀ values for Norleual and HGFwere 3.1±2.1 pM and 29.4±14.7 pM (mean ±SEM, n=3), respectively; thelatter value was similar to that reported previously (Higuchi et al.1991 Biochem. Biophys. Res. Commun. 176: 599-607). To further test thehypothesis that Norleual interacted with the c-Met system, ¹²⁵I-Norleualwas bound to HEK293 cell membranes alone or in the presence of HGF orNorleual. Both ligands competed for ¹²⁵I-Norleual specific binding toHEK293 cell membranes, and Norleual also apparently interacted withbinding sites on HEK293 membranes that were not shared with HGF (FIGS.3C and 3D). Together, these findings were consistent with a directinteraction between Norleual and c-Met or HGF.

Whether the interaction of Norleual with the c-Met receptor hadfunctional consequences regarding c-Met signaling was next investigatedby determining whether Norleual could alter the phosphorylation statusof cellular protein tyrosine phosphate (FIG. 4) and more specifically,of c-Met (i.e., c-Met activation), and also whether Norleual couldmodulate Gab1 association with c-Met. Human embryonic kidney HEK293cells were activated by HGF in the presence or absence of Norleual.Solubilized membranes were immunoprecipitated (IP) with an anti-c-Metantibody and immunoblotted (IB) for total c-Met, anti-phospho-tyrosineto detect the activated form of c-Met, and anti-Gab1 to detectc-Met/Gab1 association. FIG. 5A shows that c-Met auto-phosphorylationwas induced in a dose-dependent manner by HGF with saturation occurringat 550 pM. Norleual applied alone did not alter c-Met phosphorylation,while Norleual at 20 pM significantly reduced HGF-dependent c-Met andGab1 phosphorylation (FIGS. 5B, 5E, FIG. 6). In addition, Norleualsignificantly reduced HGF-initiated association between Gab1 and c-Met(FIGS. 5C, 5F). To confirm these findings, treated HEK293 lysates wereprecipitated with anti-Gab1 and blotted for total Gab1 and phospho-Gab1,its activated form. Again, Norleual inhibited HGF-induced Gab1phosphorylation (FIGS. 5D, 5G). Collectively, these data indicated thatthe AT₄ antagonist Norleual (Compound 2, SEQ ID NO:43) markedlyattenuated HGF-dependent c-Met activation.

Example 3 Inhibition of HGF-Induced Cell Scattering by anAngiotensin-Like Factor

To explore the physiological significance of the angiotensin-like factorNorleual's ability to depress c-Met signaling, Norleual's effect on HGFinduced cell scattering of Madin-Darby canine kidney (MDCK) cells wasassessed with the coverslip assay (Miao et al., 2003 J. Cell Biol. 162:1281-1292). See Example 1 for other Materials and Methods. Norleualinhibited HGF-induced MDCK cell scattering, yielding a response that wassimilar to controls (FIG. 8A).

Example 4 Anti-Cancer Activity of the Angiotensin-Like Factor Norleual

In this Example, anti-cancer activity of the angiotensin-like factorNorleual (Compound 2, SEQ ID NO:43) was demonstrated. See Example 1 forMaterials and Methods. Norleual was applied to HGF treated B16-F10murine melanoma cells in a scratch wound assay. HGF accelerated thewound closure, however Norleual was able to attenuate HGF stimulatedclosure six hours after scratch (FIG. 11A).

In addition, molecules associated with proliferation and migrationsignal transduction in B16-F10 melanoma cells were investigated in thescratch wound assay. HGF alone promoted enhanced phosphorylation of thesignal transduction molecules Erk1/2, an effect that was attenuated bythe presence of Norleual (FIG. 11B). Specificity of this effect ofNorleual for a signal transduction pathway in which Erk1/2 componentsare present was demonstrated by the observation that Norleual did notaffect Akt phosphorlyation that was induced by HGF (FIG. 11B). Thesedata show inhibition by Norleual of HGF-induced signaling.

Example 5 Effect of an Angiotensin-Like Factor on Angiogenesis

In this Example, anti-angiogenic activity of the angiotensin-like factorNorleual (Compound 2, SEQ ID NO:43) was demonstrated. See Example 1 forMaterials and Methods. Angiogenesis was evaluated with the ex vivo mouseaortic ring assay in the presence and absence of Norleual. Robustangiogenic sprouts were observed in control rings, while angiogenicgrowth was significantly attenuated in Norleual-treated aortic rings(FIG. 16A). Because c-Met activation promotes angiogenesis inendothelial cells (Jiang et al., 2005 Crit. Rev. Oncol. Hematol. 53:35), the effect of Norleual on c-Met signaling in endothelial cells wasinvestigated. Norleual inhibited HGF-induced Met/Gab1 association inhuman umbilical vein endothelial cells (HUVEC) in vitro (FIG. 16B).Together, these observations indicated that inhibition of angiogenesisby Norleual may be the result of attenuation by Norleual of c-Metsignaling in endothelial cells.

Example 6 In Vivo Anti-Cancer Activity of the Angiotensin-Like FactorNorleual

In this example, the effect of Norleual on in vivo tumor growth wastested. A role for c-Met signaling in B16-F10 tumor invasion andmetastasis has been reported (Ferraro, et al., 2006 Oncogene. 25:3689-3698). Materials and Methods were as described herein including inExample 1. Norleual inhibited primary B16-F0 murine melanoma growth inmale C57 mice (FIG. 13A). When the effect of the angiotensin-like factorNorleual was tested on secondary tumor growth using the in vivopulmonary B16-F10 melanoma metastasis model in mice (Fidler andNicolson, 1976 J Natl Cancer Inst. 57: 1199), striking results wereobtained. As shown in FIG. 13, Norleual dramatically inhibited B16-F10lung metastasis in mice (FIGS. 13B, 13C).

Example 7 Effect of Several Angiotensin-Like Factors on Angiogenesis

This Example shows alteration of angiogenesis by angiotensin-likefactors as provided herein, in particular, that Nle¹-AngIV, an AT₄receptor agonist; augmented the proliferation and migration of humanendothelial calls, while NORLEUAL, an AT₄ receptor antagonist, inhibitedthese processes. Materials and methods were as described in this Exampleand in Example 1.

Disc Angiogenesis Assay. Granulation tissue was experimentally inducedby implanting a polyvinyl alcohol foam disc 10 mm in diameter and 2 mmthick (Fajardo et al., 1988). A 2 mm core was removed from the center ofeach disc, saturated with 50 μg of Nle¹-AngIV (SEQ ID NO:41),Nle¹-Leu³-Ψ(CH₂NH₂)³⁻⁴-AngIV, Hexamide, or ethanol (control), air dried,then sealed using a fixative (Elvax, DuPont) to allow sustained deliveryof the drug treatment into the disc and adjacent tissues. The cores weredried for 30 min, then reinserted into the sponge discs. Impermeable topand bottom covers were prepared by coating 10 mm diameter paper discswith a plexiglass coating, then glued to the top and bottom of thesponge discs, leaving only the rim of the disc available for centripetalcellular penetration. Just prior to implantation, the discs were soakedbriefly in sterile saline.

Thirty-two Sprague Dawley female rats (225-275 gr) were used to evaluatefour compounds for angiogenic or anti-angiogenic properties using thedisc angiogenesis assay. Animals were anesthetized using sodiumpentobarbital (40 mg/kg IP). The dorsum was then shaved and asepticallyprepped. Each animal received four discs. Animals receiving treateddiscs were implanted with 2 treated discs and 2 ethanol-soaked controldiscs. Thus, in a group of 4 animals there were 8 treatment discs, and 8control discs. These control discs were used to determine whether theligand had potential systemic effects. Another group of 4 animalsreceived only ethanol-soaked control discs (spontaneous growth controlsor SGC discs) for an n=16. However, one SGC was fragmented duringprocessing leaving an n=15.

Four 2 cm cutaneous incisions were made along the dorsum (left thoracic,right thoracolumbar, left thoracolumbar, and right lumbar), andsubcutaneous pouches opened in the fascia using blunt dissection. Discswere moistened in sterile saline, inserted deep to the pouch, and theskin closed with sterile surgical staples. Animals were recovered underheat lamps until sternally recumbent, then individually housed for 14days in an AAALAC accredited facility (water and food ad libitum; 12/12hour light cycle).

After 14 days the animals were euthanized in a CO₂ chamber and the discswere removed. The outer plexiglass covers were discarded and the spongediscs were individually inserted into plastic cassettes, placed inneutral buffered formalin (10%) for 48-72 hours, followed by saturationin 70% ethanol for 24 hours, and then paraffin embedded. Six planar 3 μmsections were cut from each disc and mounted on electrostatic slides(Sigma). Slides were deparaffinized, then hydrated in ethanol ofdecreasing dilution with water (100%-50%), and then stained with 0.33mg/ml toluidine blue for 30 sec to permit visualization of the area ofthe disc penetrated by migrating cells. Three washes with waterfollowed, then dehydration by reversal of the alcohol baths. Finally,the slide was rinsed in Clear Rite 3 and coverslipped. Cellularpenetration into the discs was quantified using a digital imaging system(Imaging Research Inc., St. Catharines, Ontario, Canada). The total areaof the disc was calculated, as was the area containing stained cells.Data were then reported as total cellular area as a proportion of discarea.

Results. Following staining with toludine blue or Factor VIII,neovascularization was quantitated by densitometry. Results aresummarized in FIG. 19. A comparison of SGC discs (n=15) to treatmentdiscs indicated all treatments were anti-angiogenic at the tested doses,p<0.01 in all cases except Nle1-AngIV (SEQ ID NO:41), the lone AT4receptor agonist. Those compounds exhibiting antagonist activityincluded Nle-Tyr-Ile-His, Nle-Tyr-Ile-(6)aminohexanoic amide, andNORLEUAL.

Example 8 In Vivo Anti-Cancer Activity of the Angiotensin-Like FactorNorleual

This Example describes Norleaul effects in a mouse model of breastcancer in which animals were orthotopically injected with +SA WAZ-2Tmouse mammary carcinoma cells (5×10⁵) into the mammary fat pad of11-week old female Balb/c mice. The +SA cell line was chosen due to itsaggressive nature and ability to form highly vascularized tumors with100% incidence per injection (Danielson et al., 1986). NORLEUAL (SEQ IDNO:43) was delivered via Elvax slow release pellets at doses of 0.3mg/pellet, 0.03 mg/pellet, and 0.003 mg/pellet, implanted into themammary glands immediately prior to tumor cell injection. These pelletsprovided an estimated continuous release of NORLEUAL at doses equivalentto 37.5 μg/kg/day, 3.75 μg/kg/day, and 0.375 μg/kg/day, respectively(based on in vitro release assays of pellets, data not shown). Thismethod of drug administration was consistent with previous reportsdemonstrating increased efficacy of angiogenic inhibitors delivered on alow-dose, continuous schedule (Kisker, et al., 2001), as opposed toconventional cytotoxic chemotherapy, which is administered in a bolusinjection of the maximum tolerated dose. Tumor growth was monitoredevery 2-3 days, based on caliper measurements of the two longestdiameters and calculating tumor volume.

Control tumors typically required a 7 day lag period before a palpabletumor mass arose, a timeframe consistent with previous in vivo studiesusing this same cell line (Danielson et al., 1986). NORLEUAL treatment,however, increased the delay in onset up to 21 days (0.3 mg/pellet;n=6). In addition, NORLEUAL treatment significantly decreased tumorvolume in a dose-dependent manner, with the most effective dosedemonstrating 97% inhibition of tumor volume compared to control(control tumor volume: 389.6±107 mm³; 0.3 mg/pellet NORLEUAL-treatedtumor volume: 20.2±12 mm³, n=6, P<0.001). NORLEUAL given at 0.03mg/pellet and 0.003 mg/pellet also significantly reduced tumor volumecompared to that of control tumors (70.5±47 mm³, n=6, P<0.007 and112.7±61 mm³, n=6, P<0.02, respectively). Furthermore, control tumorsremoved at the conclusion of the experiment were visibly vascularized,while treated tumors were pale in color with a total lack of visiblevascularization. Further analysis of tumor vascularization byimmunohistochemical staining with Factor VIII antibody confirmed thereduced vascularization of treated tumors compared to that of controltumors, both in the surrounding normal mammary tissue and within thetumor mass (control tumor mean vessel count/high power field 27.8±3 vs.0.3 mg/pellet NORLEUAL-treated tumor mean vessel count/high power field3.0±0.9; n=5, P<0.0001).

Example 9 Preparation of Angiotensin-Like Factors

A library of synthetic angiotensin IV receptor ligands was constructedas candidate angiotensin-like factors, by computer modeling and rationaldrug design approaches. Briefly, acidic, basic, aromatic or branch chainamino acids were systematically substituted for each position in anangiotensin IV polypeptide. Additionally, each constituent amino acidresidue was either deleted or converted to a d-amino acid form, andpeptide bond isosteres were constructed with several amino acids.Putative agonist and antagonist molecules were modeled to yield apharmacophore structure, including comparison with known molecules atten atomic centers.

Screening of a ligand for its ability to inhibit c-Met receptorphosphorylation was tested by tyrosine kinase receptor protein array.Briefly, human umbilical vein endothelial cells (HUVECs) grown toconfluence were treated for 5 minutes with COMPOUND 2 (SEQ ID NO:43).Cells were then lysed and total protein collected. As illustrated inFIGS. 4A and 4B, the angiotensin ligand inhibited c-Met phosphorylationwhen compared with controls.

Example 10 Angiotensin-Like Factor Competes with HGF for Binding toC-Met Receptor in Cell Membranes

The ability of COMPOUND 2 (SEQ ID NO:43, Norleual) acting as a ligand tocompete with hepatocyte growth factor for binding to the c-Met receptorwas tested in human embryonic kidney cells 293 (HEK 293). See Example 1for materials and methods.

Briefly, COMPOUND 2 was iodinated with ¹²⁵I and bound and cross-linkedto HEK cell membranes with or without 1 nM COMPOUND 2 or 1 nM hepatocytegrowth factor. Following solubilization and separation of free label bymolecular sieving, total radioactive counts were determined. The resultsshowed that hepatocyte growth factor blocked ¹²⁵I-COMPOUND 2 binding toHEK cells, as presented in FIG. 3A. The ability of the ligand COMPOUND 2to compete with hepatocyte growth factor binding to the c-Met receptorwas also tested using mouse liver plasma membranes and intact HEK293cells. Briefly, mouse liver was homogenized and incubated with ¹²⁵I-HGFwith various concentrations of HGF or COMPOUND 2. Membranes werepelleted and unbound label was removed by washing, and total radioactivecounts were determined. Next, HEK293 cell membranes were cross-linkedwith ¹²⁵I-HGF with unlabeled HGF or COMPOUND 2. Results indicatedCOMPOUND 2 is a competitive inhibitor of HGF binding to the c-Metreceptor, as illustrated in FIG. 3B. The K_(D) for HGF was approximately29 pM, while the K_(D) for COMPOUND 2 was approximately 3 pM.

Example 11 Angiotensin-Like Factor Alters Gene Expression in EndothelialCells

Angiotensin-like factors as candidate c-Met receptor ligands were testedfor the ability to modulate expression of extracellular matrix-relatedproteins in human umbilical vein endothelial cells (HUVECs) by genemicroarray. Briefly, HUVECs were treated for 8 days in the presence orabsence of test compounds at a concentration of 1 pM; Norleual (Compound2, SEQ ID NO:43) was used as an exemplary angiotensin-like factor. RNAwas harvested and transcriptional activities of 96 extracellular matrixrelated genes were assessed using gene microarrays. Gene expressionlevels that differed from the levels in control samples by at least twofold for cells treated with Compound 2 included: CD44, catenin, cateninβ, catenin δ1, MMP-1, PAI-1, thrombospondin 1, and integrin α-2.

Example 12 Norleual Alteration of Angiogenesis-Related Gene Expression

Angiotensin-like factor Norleual (COMPOUND 2) was tested for its abilityto modulate angiogenic genes in murine mammary tumors. Briefly, Balb/cmice grafted with +SA murine mammary cancer cells were treated withCompound 2. After 28 days of intraperitoneal administration via anosmotic pump (at a dose of 1 mg/kg/day), primary tumors were harvestedand RNA was collected. Transcriptional activities of 96angiogenesis-related genes were assessed using gene microarrays. Genesthat differed from control by at least five-fold included: Adamts 1,CD36, Connective tissue growth factor, PECAM 1, Cxcl 4, restin, Ccl 2,TNF-α, VEGF-α, and VEGF-β.

Example 13 Angiotensin-Like Factor Alteration of Cell Scattering

The ability of an angiotensin-like factor, Norleual (COMPOUND 2), toinhibit hepatocyte growth factor-dependent functions, includingproliferation, cell scattering, and c-Met mediated collagen I invasionin Madin-Darby canine kidney (MDCK) cells was tested using methodologiesdescribed in Example 1. Briefly, MDCK cells were grown to confluence,and COMPOUND 2 and/or heptocyte growth factor were added as appropriate.For the proliferation assay, hepatocyte growth factor was used at 10ng/ml, and COMPOUND 2 was used at 10⁻⁶M, 10⁻⁸M, 10⁻¹⁰M, 10⁻¹²M, or10⁻¹⁴M. For the scattering assay, hepatocyte growth factor was used at20 ng/ml, and COMPOUND 2 was used at 10⁻¹⁰M. For the collagen assay,hepatocyte growth factor was used at 20 ng/ml, and COMPOUND 2 was usedat 10⁻¹⁰M, 10⁻¹¹M, or 10⁻¹²M. For all assays, cells were fixed inmethanol and stained with Diff-Quick Wright-Giemsa (Dade-Behring). Asillustrated in representative FIG. 7, COMPOUND 2 inhibited hepatocytegrowth factor-dependent cell scattering. Compound 2 also inhibitedcellular proliferation, and c-Met mediated collagen I invasion by MDCKcells.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for altering a hepatocyte growth factor activity or a c-Metreceptor activity in one cell or in a plurality of cells, comprising:contacting (i) a cell or plurality of cells with (ii) a composition thatcomprises an isolated angiotensin-like factor, under conditions and fora time sufficient for the angiotensin-like factor to interact with acell surface c-Met receptor, wherein the angiotensin-like factor iscapable of specifically binding to the cell surface c-Met receptor, andthereby altering hepatocyte growth factor activity or c-Met receptoractivity.
 2. The method of claim 1 wherein the angiotensin-like factorcomprises a composition that is selected from the group consisting of:(a) a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof,of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is phenylalanine,tryptophan or tyrosine, X₂ is isoleucine, leucine, alanine, valine,phenylalanine, proline, methionine or tryptophan, and X₃ is lysine,arginine or histidine; (b) a polypeptide of no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is a natural ornon-natural amino acid having an aromatic side chain, X₂ is a natural ornon-natural amino acid having a hydrophobic side chain, and X₃ is anatural or non-natural amino acid having a basic side chain; (c) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, saidpolypeptide comprising (i) a tripeptide having an amino acid sequencethat is selected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and

(ii) at least one of an amino terminus and a carboxy terminus, each ofsaid amino terminus and said carboxy terminus consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; (d) thepolypeptide or peptidomimetic of (c) which comprises the amino acidsequence Nle-Tyr-Leu-Ψ-His-Pro-Phe as set forth in SEQ ID NO:43, whereinΨ consists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II] (e) the polypeptide or peptidomimetic of (b) whichcomprises at least one amino acid sequence selected from the groupconsisting of (SEQ ID NO:9) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu,(SEQ ID NO:25) Arg-Val-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:33)Val-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:41) Nle-Tyr-Ile-His-Pro-Phe, (SEQ IDNO:42) Val-ψ-Tyr-Leu-ψ-His-Pro-Phe, (SEQ ID NO:45)Nle-ψ-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:46) Leu-ψ-Tyr-Leu-ψ-His-Pro-Phe,(SEQ ID NO:47) Nle-Tyr-Ile-His, (SEQ ID NO:48)Nle-Tyr-Ile-(CH₂)₆-Phe-amide, (SEQ ID NO:49) Nle-Tyr-Ile-Sar-Sar-dPhe,(SEQ ID NO:50) Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val- Tyr,(SEQ ID NO:52) Nle-Tyr-Ile-6(amino) hexanoic acid amide, (SEQ ID NO:53)Nle-Tyr-Ile-His-Pro, (SEQ ID NO:54) Lys-Tyr-Ile-His-Pro-Phe, (SEQ IDNO:55) benzyl-cysteine-Tyr-Ile-His-Pro-Phe, (SEQ ID NO:56)dNle-Tyr-Ile-His-Pro-Phe, [SEQ ID NO:57] Nle-Tyr-Ile-ψ-His-Pro-Phe, [SEQID NO:58] Nle-Tyr-Val-ψ-His-Pro-Phe, [SEQ ID NO:59]Nle-Tyr-Nle-ψ-His-Pro-Phe, [SEQ ID NO:60] Nle-Phe-Leu-ψ-His-Pro-Phe,[SEQ ID NO:61] Nle-Phe-Ile-ψ-His-Pro-Phe, [SEQ ID NO:62]Nle-Phe-Val-ψ-His-Pro-Phe, [SEQ ID NO:63] Nle-Phe-Nle-ψ-His-Pro-Phe,[SEQ ID NO:64] Nle-Tyr-Leu-ψ-Arg-Pro-Phe, [SEQ ID NO:65]Nle-Tyr-Ile-ψ-Arg-Pro-Phe, [SEQ ID NO:66] Nle-Tyr-Val-ψ-Arg-Pro-Phe,[SEQ ID NO:67] Nle-Tyr-Nle-ψ-Arg-Pro-Phe, [SEQ ID NO:68]Nle-Phe-Leu-ψ-Arg-Pro-Phe, [SEQ ID NO:69] Nle-Phe-Ile-ψ-Arg-Pro-Phe,[SEQ ID NO:70] Nle-Phe-Val-ψ-Arg-Pro-Phe, [SEQ ID NO:71]Nle-Phe-Nle-ψ-Arg-Pro-Phe, [SEQ ID NO:72] Nle-Tyr-Leu-ψ-Lys-Pro-Phe,[SEQ ID NO:73] Nle-Tyr-Ile-ψ-Lys-Pro-Phe, [SEQ ID NO:74]Nle-Tyr-Val-ψ-Lys-Pro-Phe, [SEQ ID NO:75] Nle-Tyr-Nle-ψ-Lys-Pro-Phe,[SEQ ID NO:76] Nle-Phe-Leu-ψ-Lys-Pro-Phe, [SEQ ID NO:77]Nle-Phe-Ile-ψ-Lys-Pro-Phe, [SEQ ID NO:78] Nle-Phe-Val-ψ-Lys-Pro-Phe,[SEQ ID NO:79] Nle-Phe-Nle-ψ-Lys-Pro-Phe, [SEQ ID NO:80] γ-aminobutyricacid-Tyr-Ile, and [SEQ ID NO:81] β-Ala-Tyr-Ile,

wherein Ψ consists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II].
 3. The method of claim 1 wherein the angiotensin-likefactor comprises an antibody, or an antigen-binding fragment of saidantibody, that competitively inhibits binding of a native HGF hingeregion polypeptide to the cell surface c-Met receptor, said native HGFhinge region polypeptide comprising the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82].
 4. The method of claim 3wherein the antibody is a monoclonal antibody.
 5. The method of claim 3wherein the antibody is a chimeric antibody or a humanized antibody. 6.The method of claim 3 wherein the antigen-binding fragment is selectedfrom the group consisting of a Fab fragment, a Fab′ fragment, a (Fab′)₂fragment, an Fd fragment, an Fv fragment and an scFv.
 7. The method ofclaim 3 wherein the antibody comprises an anti-idiotype antibody thatspecifically recognizes a complementarity determining region of animmunoglobulin that specifically binds to the native HGF hinge regionpolypeptide.
 8. The method of any one of claims 1 to 3 wherein theangiotensin-like factor competitively inhibits binding of native HGF tothe cell surface c-Met receptor.
 9. The method of claim 1 wherein thestep of contacting is selected from the group consisting of contactingin vivo and contacting ex vivo.
 10. The method of any one of claims 1-3wherein the angiotensin-like factor is glycosylated.
 11. The method ofclaim 1 wherein the hepatocyte growth factor activity or the c-Metreceptor activity comprises at least one activity that is selected fromthe group consisting of (i) induction of cellular proliferation, (ii)induction of cell scattering or migration, and (iii) alteration of ac-Met receptor pathway component phosphorylation state.
 12. The methodof claim 11 wherein alteration of a c-Met receptor pathway componentphosphorylation state comprises induction of Gab1 activation.
 13. Themethod of either claim 1 or claim 2 wherein the angiotensin-like factorcomprises a peptidomimetic and wherein the peptidomimetic comprises atleast one moiety that is selected from the group consisting of anon-natural amino acid residue and a reduced peptide bond.
 14. Themethod of claim 1 wherein the cell or plurality of cells comprises atissue or an organ.
 15. The method of claim 1, wherein the hepatocytegrowth factor activity or c-Met receptor activity in the cell isdecreased subsequent to the step of contacting, relative to thehepatocyte growth factor activity or c-Met receptor activity in the cellprior to the step of contacting.
 16. The method of claim 15 wherein thehepatocyte growth factor activity or c-Met receptor activity comprisesan activity that is selected from the group consisting of (i) inductionof cell proliferation, (ii) induction of cell migration, (iii) inductionof extracellular matrix disruption, (iv) induction of dysregulation ofapoptosis, (v) induction of cellular extravasation, (vi) induction ofaltered expression of an adhesion molecule that is selected from thegroup consisting of CD44, catenin, catenin β, catenin δ1, MMP-1, PAI-1,thrombospondin 1, and integrin α-2, and (vii) induction of alteredexpression of an angiogenesis-related molecule that is selected from thegroup consisting of: Adamts 1, CD36, Connective tissue growth factor,Pecam 1, Cxcl 4, restin, Ccl 2, TNF-α, VEGF-α, and VEGF-β.
 17. Themethod of claim 1, wherein the hepatocyte growth factor activity orc-Met receptor activity is increased in the cell subsequent to the stepof contacting, relative to the hepatocyte growth factor activity orc-Met receptor activity in the cell prior to the step of contacting. 18.The method of claim 17 wherein the hepatocyte growth factor activity orc-Met receptor activity comprises an activity that is selected from thegroup consisting of (i) induction of angiogenesis, (ii) induction ofneurite growth or axon guidance, (iii) induction of celldifferentiation, (iv) induction of bone regeneration, and (v) inductionof tissue repair.
 19. A method of altering a hepatocyte growth factoractivity or a c-Met receptor activity in a subject, comprisingadministering to the subject a composition that comprises an isolatedangiotensin-like factor, under conditions and for a time sufficient forthe angiotensin-like factor to interact with a cell surface c-Metreceptor in the subject, wherein the angiotensin-like factor is capableof specifically binding to the cell surface c-Met receptor, and therebyaltering a hepatocyte growth factor activity or a c-Met receptoractivity in the subject.
 20. A method of treating or preventing acondition associated with c-Met dysregulation in a subject, comprisingadministering to the subject a composition that comprises an isolatedangiotensin-like factor, under conditions and for a time sufficient forthe angiotensin-like factor to interact with a cell surface c-Metreceptor in the subject, wherein the angiotensin-like factor is capableof specifically binding to the cell surface c-Met receptor, and therebytreating or preventing the condition associated with c-Metdysregulation.
 21. The method of claim 20 wherein the conditionassociated with c-Met dysregulation is selected from the groupconsisting of (i) cellular hyperproliferation, (ii) inflammation, (iii)an increased level of angiogenesis relative to the level of angiogenesisin a control subject known to be free of a risk for having a conditionassociated with c-Met dysregulation, (iv) an increased level of adiposedeposition relative to the level of adipose deposition in a controlsubject known to be free of a risk for having a condition associatedwith c-Met dysregulation, and (v) cognitive dysfunction.
 22. The methodof either claim 19 or claim 20 wherein the angiotensin-like factor isselected from the group consisting of (a) a polypeptide of no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 aminoacids, or a peptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is phenylalanine,tryptophan or tyrosine, X₂ is isoleucine, leucine, alanine, valine,phenylalanine, proline, methionine or tryptophan, and X₃ is lysine,arginine or histidine; (b) a polypeptide of no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is a natural ornon-natural amino acid having an aromatic side chain, X₂ is a natural ornon-natural amino acid having a hydrophobic side chain, and X₃ is anatural or non-natural amino acid having a basic side chain; (c) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, saidpolypeptide comprising (i) a tripeptide having an amino acid sequencethat is selected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and

(ii) at least one of an amino terminus and a carboxy terminus, each ofsaid amino terminus and said carboxy terminus consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; (d) thepolypeptide or peptidomimetic of (c) which comprises the amino acidsequence Nle-Tyr-Leu-Ψ-His-Pro-Phe as set forth in SEQ ID NO:43, whereinΨ consists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II]; and (e) the polypeptide or peptidomimetic of (b) whichcomprises at least one amino acid sequence selected from the groupconsisting of SEQ ID NOS:9, 25, 33, 41, 42, 45-50, 52-80 and
 81. 23. Themethod of either claim 19 or claim 20 wherein the angiotensin-likefactor comprises an antibody, or an antigen-binding fragment of saidantibody, that competitively inhibits binding of a native HGF hingeregion polypeptide to the c-Met receptor, said native HGF hinge regionpolypeptide comprising the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82].
 24. The method of claim 23wherein the antibody is a monoclonal antibody.
 25. The method of claim23 wherein the antibody is a chimeric antibody or a humanized antibody.26. The method of claim 23 wherein the antigen-binding fragment isselected from the group consisting of a Fab fragment, a Fab′ fragment, a(Fab′)₂ fragment, an Fd fragment, an Fv fragment and an scFv.
 27. Themethod of claim 23 wherein the antibody comprises an anti-idiotypeantibody that specifically recognizes a complementarity determiningregion of an immunoglobulin that specifically binds to the native HGFhinge region polypeptide.
 28. The method of either claim 19 or claim 20wherein the angiotensin-like factor competitively inhibits binding ofnative HGF to the c-Met receptor.
 29. The method of claim 22 wherein theangiotensin-like factor is glycosylated.
 30. The method of claim 22further comprising administering to the subject, in addition to theangiotensin-like factor, a second composition that comprises atherapeutic agent.
 31. The method of claim 30 wherein the therapeuticagent is selected from the group consisting of (a) a chemotherapeuticagent, (b) a nucleic acid disrupting agent, (c) an anti-proliferativeagent, (d) a radiotherapy compound, (e) a cytotoxic agent, (f) ananti-inflammatory agent, (g) a statin, (h) a pro-angiogenesis agent, (i)a chemoattractant, and (j) any combination of two or more of (a)-(i).32. A method of diagnosing a subject having, suspected of having orsusceptible to a condition associated with c-Met receptor dysregulation,comprising: (a) contacting (i) a first biological sample from a firstsubject having, suspected of having or susceptible to a conditionassociated with c-Met receptor dysregulation, which sample comprises acell surface c-Met receptor, and (ii) an isolated angiotensin-likefactor, under conditions and for a time sufficient for theangiotensin-like factor to interact with the cell surface c-Metreceptor, wherein the angiotensin-like factor is capable of specificallybinding to the cell surface c-Met receptor; and (b) determining analtered level of cell surface c-Met receptor in the first biologicalsample, relative to the level of cell surface c-Met receptor in a secondbiological sample obtained from a second subject known to be free ofrisk for having the condition associated with c-Met receptordysregulation.
 33. The method of claim 32 wherein the angiotensin-likefactor is selected from the group consisting of: (a) a polypeptide of nomore than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4or 3 amino acids, or a peptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is phenylalanine,tryptophan or tyrosine, X₂ is isoleucine, leucine, alanine, valine,phenylalanine, proline, methionine or tryptophan, and X₃ is lysine,arginine or histidine; (b) a polypeptide of no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is a natural ornon-natural amino acid having an aromatic side chain, X₂ is a natural ornon-natural amino acid having a hydrophobic side chain, and X₃ is anatural or non-natural amino acid having a basic side chain; (c) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, saidpolypeptide comprising (i) a tripeptide having an amino acid sequencethat is selected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and

(ii) at least one of an amino terminus and a carboxy terminus, each ofsaid amino terminus and said carboxy terminus consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; (d) thepolypeptide or peptidomimetic of (c) which comprises the amino acidsequence Nle-Tyr-Leu-Ψ-His-Pro-Phe as set forth in SEQ ID NO:43, whereinΨ consists of a reduced peptide bond of formula II:—CH₂—NH₂—  [II]; and (e) the polypeptide or peptidomimetic of (b) whichcomprises at least one amino acid sequence selected from the groupconsisting of SEQ ID NOS:9, 25, 33, 41, 42, 45-50, 52-80 and
 81. 34. Themethod of claim 32 wherein the angiotensin-like factor comprises anantibody, or an antigen-binding fragment of said antibody, thatcompetitively inhibits binding of a native HGF hinge region polypeptideto the c-Met receptor, said native HGF hinge region polypeptidecomprising the amino acid sequence Lys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ IDNO:82].
 35. The method of claim 34 wherein the antibody is a monoclonalantibody.
 36. The method of claim 34 wherein the antibody is a chimericantibody or a humanized antibody.
 37. The method of claim 34 wherein theantigen-binding fragment is selected from the group consisting of a Fabfragment, a Fab′ fragment, a (Fab′)₂ fragment, an Fd fragment, an Fvfragment and an scFv.
 38. The method of claim 34 wherein the antibodycomprises an anti-idiotype antibody that specifically recognizes acomplementarity determining region of an immunoglobulin thatspecifically binds to the native HGF hinge region polypeptide.
 39. Themethod of claim 33 wherein the angiotensin-like factor is glycosylated.40. The method of claim 32 wherein the condition associated with c-Metdysregulation is selected from the group consisting of cellularhyperproliferation, inflammation, reduced angiogenesis, adiposedeposition and cognitive dysfunction.
 41. An antibody, or anantigen-binding fragment of said antibody, that competitively inhibitsbinding of a native hepatocyte growth factor (HGF) hinge regionpolypeptide to the c-Met receptor, said native HGF hinge regionpolypeptide comprising the amino acid sequenceLys-Asp-Tyr-Ile-Arg-Asn-Cys [SEQ ID NO:82].
 42. The antibody of claim 41that is a monoclonal antibody.
 43. The antibody of claim 41 wherein theantibody is a chimeric antibody or a humanized antibody.
 44. Theantibody of claim 41 wherein the antigen-binding fragment is selectedfrom the group consisting of a Fab fragment, a Fab′ fragment, a (Fab′)₂fragment, an Fd fragment, an Fv fragment and an scFv.
 45. The antibodyof claim 41 wherein the antibody comprises an anti-idiotype antibodythat specifically recognizes a complementarity determining region of animmunoglobulin that specifically binds to the native HGF hinge regionpolypeptide.
 46. The antibody of claim 41 wherein the antibody iscapable of altering at least one HGF activity or c-Met receptoractivity.
 47. The antibody of claim 46 wherein the hepatocyte growthfactor activity or c-Met receptor activity comprises at least oneactivity that is selected from the group consisting of (i) induction ofcellular proliferation, (ii) induction of cell scattering or migration,and (iii) alteration of a c-Met receptor pathway componentphosphorylation state.
 48. The antibody of claim 47 wherein alterationof a c-Met receptor pathway component phosphorylation state comprisesinduction of Gab1 activation.
 49. A composition that alters a hepatocytegrowth factor activity or a c-Met receptor activity in a cell or in aplurality of cells, comprising a polypeptide or a peptidomimetic thereofthat does not comprise the polypeptide sequence set forth in either oneof SEQ ID NOS:43 or 54 and that is selected from the group consistingof: (a) a polypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimeticthereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is phenylalanine,tryptophan or tyrosine, X₂ is isoleucine, leucine, alanine, valine,phenylalanine, proline, methionine or tryptophan, and X₃ is lysine,arginine or histidine; (b) a polypeptide of no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is a natural ornon-natural amino acid having an aromatic side chain, X₂ is a natural ornon-natural amino acid having a hydrophobic side chain, and X₃ is anatural or non-natural amino acid having a basic side chain; (c) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, saidpolypeptide comprising (i) a tripeptide having an amino acid sequencethat is selected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and

(ii) at least one of an amino terminus and a carboxy terminus, each ofsaid amino terminus and said carboxy terminus consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; and (d)the polypeptide or peptidomimetic of (b) which comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NOS:9,25, 33, 41, 42, 45-50, 52, 53, 55-80 and
 81. 50. A pharmaceuticalcomposition comprising: (a) a polypeptide that comprises an amino acidsequence selected from the group consisting of the sequence set forth inSEQ ID NO:47 and the sequence set forth in SEQ ID NO:52, or apeptidomimetic thereof; and (b) a pharmaceutically acceptable carrier.51. A pharmaceutical composition comprising a polypeptide, or apeptidomimetic thereof; and a pharmaceutically acceptable carrier,wherein the polypeptide or peptidomimetic does not comprise thepolypeptide sequence set forth in either one of SEQ ID NOS:43 or 54 andis selected from the group consisting of: (a) a polypeptide of no morethan 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3amino acids, or a peptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is phenylalanine,tryptophan or tyrosine, X₂ is isoleucine, leucine, alanine, valine,phenylalanine, proline, methionine or tryptophan, and X₃ is lysine,arginine or histidine; (b) a polypeptide of no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids, or apeptidomimetic thereof, of general formula I:N—X₁—X₂—X₃—C  [I] wherein: N is an amino terminus of the peptide orpeptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16 or 17 amino acids that are independently selected fromnatural and non-natural amino acids, C is a carboxy terminus of thepeptide or peptidomimetic and consists of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 or 17 amino acids that are independentlyselected from natural and non-natural amino acids, X₁ is a natural ornon-natural amino acid having an aromatic side chain, X₂ is a natural ornon-natural amino acid having a hydrophobic side chain, and X₃ is anatural or non-natural amino acid having a basic side chain; (c) apolypeptide of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4 or 3 amino acids, or a peptidomimetic thereof, saidpolypeptide comprising (i) a tripeptide having an amino acid sequencethat is selected from the group consisting of: Lys-Asp-Tyr, Leu-Asp-Tyr,Asp-Tyr-Ile, Tyr-Ile-Arg, Ile-Arg-Asn, and Arg-Asn-Cys, and

(ii) at least one of an amino terminus and a carboxy terminus, each ofsaid amino terminus and said carboxy terminus consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids that areindependently selected from natural and non-natural amino acids; and (d)the polypeptide or peptidomimetic of (b) which comprises at least oneamino acid sequence selected from the group consisting of SEQ ID NOS:9,25, 33, 41, 42, 45-50, 52, 53, 55-80 and 81.